An integrated approach towards characterising the functional mechanics and energetics of insect fight muscles

Insects are the most diverse and economically important classes of animals on earth and there is little doubt that one of the reasons for their great success is their incredible flying ability, which typically far surpasses that of vertebrates in terms of manoeuvrability and control. In the time that it takes a human to blink, a blowfly can beat its wings 50 times, powering and controlling each wingbeat using numerous tiny muscles - some as thin as a human hair. Unlike their flying verterbrate counterparts, insect wings contain no muscles; instead they are all hidden inside the thorax. Understanding how insects efficiently meet the high-energy demands of flight, using their remarkably complex flight motor therefore presents an exciting challenge that is of interest to both biologists and engineers.

In muscles there is typically a trade-off between force production and speed, which presents a problem for insect flight. Some insect orders they have evolved power muscles that do not require neuronal activation for each contraction. These power muscles can operate at high frequencies (up to 1000 Hz in some mosquitoes), while still producing high forces as the majority of the muscle can consist of contractile apparatus. However, a consequence is that these power muscles are unable to produce rapid changes in their force production, necessary for manoeuvring flight. Instead, a myriad of small steering muscles are responsible for producing rapid changes in wing motion. Nonetheless, these steering muscles must also operate at high frequencies, while simultaneously dealing with the high power output produced by the much larger power muscles.

The aim of this grant is to use an integrated approach to gain the most detailed understanding of the mechanics, function and energetics of insect flight muscle to date. We will determine how the movements, neural stiumulation, force production and efficiency of different flight muscles changes according to their role in the flight. We will compare muscles across dipteran (flies) species and other insect orders to further understand how natural selection has shaped the flight motor in species with different aerial behaviour and ecologies.

Measuring all of the above parameters simultaneously in insects is currently impossible, largely due to size limitations. Instead we will use a combination of methods to measure each separately and then combine the information to give a detailed picture of muscle function. We use time-resolved microtomography, a recently developed technique that makes it possible, for the first time, to visualise and measure the movements of the internal structures inside a live, flying insect and electrophysiology to record in vivo muscle action potential. We also record wing movements allowing us to collate data across experiments by matching wingbeat parameters.

These data will then be applied to in vitro work loop studies of the same muscles so that their length changes and neuronal activation can be simulated as if they were in vivo. This will allow us to determine how the muscles make use of mechanisms such as negative work in the steering muscles and elastic storage in the power muscles to increase efficiency and performance. We will also determine how the muscle's energy consumption contributes to the overall energetic cost of flight and flight manoeuvres, by measuring the rates of oxygen consumption and carbon dioxide production during tethered flight in a wind tunnel.

Using an integrated approach such as this we will provide a unique insight into the functional mechanisms underlying the control and energetics of insect flight. This research will be of interest to biologists interested in how natural selection alters the function of muscles adapted for different purposes. The outputs will also provide engineers inspiration for the design of flapping unmanned air systems that are typically limited by inefficient motors.

Our aim is to use an integrated approach to produce the most detailed understanding of the mechanics, function and efficiency of insect flight muscles. Key objectives we aim to answer are: how do the tiny steering muscles control the wingbeat by efficiently managing the power produced by the much larger power muscles? To what extent do the flight muscles make use of elastic storage and how does muscle and whole organism efficiency change during manoeuvres? How do the functional mechanics and efficiency of the flight muscles change across insect species and orders?

We will measure in vivo muscle strains using synchrotron-based, time-resolved microtomography; in vivo muscle action potentials using electrophysiology; and whole organism in vivo energetics using respirometry. Across all in vivo experiments we will record and calculate 3D wing kinematics using high-speed cameras. This will allow us to determine how the measured parameters change with different wingbeats, and crucially to synchronise measurements across experiments by matching wingbeats. The in vivo muscle measurements will be used to simulate in vivo muscle oscillations using the in vitro work loop approach to calculate muscle work, elastic storage and efficiency.

We will initially use blowflies (Calliphora vicinia) due to its appropriate size for the different experimental setups and the large body of time-resovled microtomography data already collected by the PI. However, we will expand to other dipteran species and insect orders to allow a comparative approach. We will aim to understand how differences in the functional mechanics of the flight muscles across species relates to differences in their flight behaviour, such as hovering in hoverflies, something that Calliphora cannot do. We will also determine how muscle mechanics and flight efficiency changes in insect orders with low wingbeat frequencies but asynchronous or synchronous flight muscles.

IMPACT SUMMARY
Obtaining an integrative understanding of the means by which insects control their flight and attain their high manoeuvrability is of broad scientific relevance and will have impact on the aeronautic industry, the general public and on the researchers employed on the grant, in addition to the benefits to the academic community (see Academic Beneficiaries).

APPLIED LINKS WITH THE POTENTIAL TO BENEFIT INDUSTRY, IMPROVE HEALTH AND DEVELOP THE 3Rs
Our insights into the mechanisms and energetics underlying the control of the wingstroke and their relationship to flight manoeuvres will be important to engineers developing autonomous unmanned air systems (UAS) for exploration, surveillance and rescue work in situations where manned flights could be unsafe or expensive. Engineers are adopting a bio-inspired approach to the design of UAS and knowledge about how the muscular and sensory systems are efficiently integrated in insects, will guide design optimization in these devices. The UK has been at the forefront of advances in our understanding insect flight aerodynamics since the pioneering work of Weis-Fogh and Ellington (University of Cambridge) and more recent work by the Oxford Animal Flight Group. Our research will help to maintain and promote the UK as a leader in insect flight research and make the UK an attractive prospect for UAS development funding.

The knowledge gained in this project will help in the development and refinement of computational models of muscle contraction. Our work is focused on how healthy muscle tissue works, which is central to developing an understanding of malfunctions that occur during ageing and disease. In all modes of locomotion, energetics and locomotor performance are linked via an energy transduction chain. Therefore, whilst our work is on flying insects the models developed should be generally applicable to other modes of locomotion and the development of an understanding of locomotor energetics in the field. Developing accurate models of muscle contraction may allow some animal experiments to be replaced and in other cases refined or reduced as simulations may allow research efforts involving animal research to be better designed.

IMPACT ON THE GENERAL PUBLIC
Animal locomotion is a topic that consistently arouses public interest. We are committed to using our research to inspire young audiences to take an interest in science. Our work will have a positive impact by informing the general public about technological advances in science and the applications of biological research. We will engage with the public through open lectures, school visits and exhibitions at museums in Oxford and Leeds. We will also apply for the Royal Society Summer Science exhibit, which attracts over 14,000 people, including 2000 school students.

OTHER SPECIFIC IMPACTS
Specific beneficiaries include the two PDRAs who will develop their scientific careers with BBSRC funding. They will be involved in a research project that crosses discipline boundaries in biology. They will benefit from working closely with laboratories in two different leading institutions (as verified by the 2014 REF). The research will also impact on the training of undergraduates who will benefit from carrying out final year research projects within our laboratories.