Invited resubmission: the Drosophila circadian clock under simulated natural conditions

Like humans, the fruitfly, Drosophila melanogaster, has an internal 24 hour clock that times its behaviour and physiology. The genetic basis for this clock is practically the same for both organisms, which also have dedicated neurons in the brain in which these clock genes are expressed. These neurons generate rhythmic signals that are transmitted to other regions of the brain and serve to generate rhythmic behaviour. These similarities between humans and flies means that the latter serve as a cheap and convenient model system for studying the molecular neurobiology of clocks. As clocks are important for human health and well-being (consider chronic shift workers, who have higher levels of health-related problems than the general population), clock research in flies has significant medical relevance.

Much of what we know about the fly clock has been collected in artificial laboratory environments at constant warm temperatures, usually in 12 hours of light followed by 12 hours of darkness, or in constant light or constant dark. While it is necessary to simplify the complex natural environment, we also run the risk of misunderstanding how clocks might work in nature. We have spent more than three years studying fly locomotor rhythms in the wild, and our results suggest that some of our cherished concepts about the fly clock, might need some serious revision. However, we cannot expect our colleagues to go through the pain of studying fly behaviour in the wild, as it is very inconvenient, difficult, and unpredictable in terms of the elements, which have us at their mercy. Thus, we need to simulate natural conditions in the laboratory, and this is what our proposal is about. We have amassed an enormous amount of field data on fly behaviour, for different fly strains, clock mutants, as well as associated meteorological observations, but we need to realistically mimic any kind of day, hot, cold, bright, overcast, with moonlight, without, in any season. In order to do this, we have developed a simulated programmable light-source which can mimic the natural light cycle in almost any environmental situation, including moonlight, twilights, and the daily changes in the spectrum. This is placed in a programmable incubator that can similarly simulate any environmental temperature cycle. In preliminary studies we have shown that we can routinely replicate almost all of the unexpected findings we have recorded in the wild, so we know our simulations are realistic. We now have an unique opportunity to study a complex behaviour in a quasi-natural setting and we can now attempt to systematically dissect and manipulate the clock, behaviourally, genetically, and neurobiologically, using the sophisticated fly-specific tools we have at our disposal, and understand more completely how natural environmental signals tune up biological rhythms.
Natural circadian entrainment has been a fundamental feature of the evolution of life and has guided the rhythmic behaviour and physiology of higher organisms. In recent centuries, natural entrainment in humans has been replaced by artificial entrainment, with ample evidence for maladaptive physiological and psychological responses. By assessing which behavioural, molecular and neurobiological features are disturbed by manipulating these external signals, our results will be of considerable interest to the scientific and lay community.

The Drosophila clock has been studied for 40 years at behavioural, molecular and neurogenetic levels, exclusively in laboratory environments. While this has been necessary to dissect the molecular framework of the clock, it raises the possibility that we might be misunderstanding how the clock works under more realistic conditions. Over the past few years we have studied the fly locomotor cycle in nature, and have amassed an enormous amount of data on wild-types and clock mutants at two locations in northern and southern Europe. Our results suggest that some of our adaptive interpretations for fly clocks are incorrect, particularly the ideas and concepts of 'anticipation' of dawn and dusk, the midday siesta under hot conditions, and the role of moonlight and social interactions in modulating circadian behaviour. We also observe some expression patterns of PER and TIM proteins that are completely unexpected.
We have simulated natural conditions closely in the laboratory, and preliminary experiments reveal that we can mimic the behavioural phenotypes we see in nature. We plan to perform a systematic analysis of the role of natural environmental cycles on behaviour and clock gene expression. We shall manipulate the natural light and temperature cycles and study the behavioural responses and longevities of both wild-type and mutants, followed by a neurogenetic dissection, initially by ablation, of key clock neuronal clusters, to examine if natural entrainment is dependent on these specific elements. We shall also study how entrainment mutants, singly and in combination, disrupt natural rhythmic behaviour in order to identify any residual entrainment that could be mediated by modalities other than temperature or light perception.

In the longer term, the main beneficiaries of our work from outside the immediate circle of chronobiologists, will be the medical profession, pharmaceutical industry and the agricultural community that is interested in insect control. The realisation that weak entrainment leads to psychological and physiological malaise in humans and animals is gathering momentum in medicine and veterinary science. However, this also impacts on policy makers who are concerned about '24-7' lifestyles and may realise that putting clocks forward or back during the year may be detrimental to overall well-being, at least for a few days, with associated economic costs, or that the school day beginning too early in the morning may not be optimal for cognitive performance. Entrainment is important in the workplace for alertness and efficiency, so there are ergonomic implications for the design of the workplace to resonate with natural entrainment. Thus the benefits of our work for health and wealth creation are quite clear, and impinge on the quality of life by generating environments conducive to creativity and efficiency.
The public are also very curious and responsive to issues concerning the body clock, and we are fortunate to have within the Genetics Department, a national CETL (Centre of Excellence in Teaching and Learning) for GENIE (Genetics, Education, Networking, Innovation and Excellence), run by Prof Annette Cashmore of our department. A significant activity of GENIE is therefore outreach, and its website attract 22,000 hits per month (http://www.le.ac.uk/genetics/genie/?searchterm=cetl). Within this website is a Virtual Genetics Education Centre for schools and colleges, higher education centres, the general public, as well as health professionals and policymakers. GENIE conducts about 35 meetings/workshops per year, and while we (CPK and ER) have contributed to GENIE functions on an ad hoc basis regularly. Consequently, we and our postdoc on this BBSRC grant shall,

1. work with the GENIE lecturers to create a section on BIORHYTHMS, GENES and HEALTH that provides basic information about the background for our research on the GENIE website. This will be targeted at the groups outlined above
2. create an educational video on SLEEP and RHYTHMS for the website
3. There will be many opportunities after mid-2011, organised by GENIE, to present our work to various groups, both local and national. We shall be particularly keen to present to health professionals.

While policymakers might take note of our results, empowering the public by educating them on the benefits of natural circadian entrainment, allows them to modify their own lifestyle accordingly. Perhaps this is the greatest tangible benefit we can provide through public communication.

Finally, we have built a prototype natural light simulator with engineering colleagues in Italy. We believe that once our work is published, that many laboratories will wish to buy one. For example, the recent EUCLOCK consortium (EC), of which we were members spent >100KE to try and develop one using a company in Germany, but it failed, as this is not trivial. Ours works, so there is scope for marketing, providing another metric for Impact.