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New directions for studying circadian biology in the wild

Final Report Summary - WILDCLOCKS (New directions for studying circadian biology in the wild)

WildClocks research, supported by the CIG grant, is at the heart of my research interests. This research combines my training in classical chronobiology with modern, mostly laboratory-based circadian methods and with concepts and methods from ecology and wildlife studies. The overarching goal of the research is in understanding the function of biological rhythms in the real world for which they have evolved. The challenge of this work is to re-connect the above-mentioned fields in terms of methods, approaches, and concepts. My research uses birds as a model taxon because they are conspicuous by sight and sound and are diurnal like humans. These features has made them a continuing central study group in the field of ecology. Birds were also originally well-studied in chronobiology but are now largely overlooked in research on biological rhythms, which has led to a scarcity of frontline methods applicable to birds (for example detailed knowledge on clock genes, lack of molecular assays etc.).

Because of this neglect, and the need to reconnect a number of different fields, the WildClocks project initially was mostly concerned with developing methods. Because it was a Career Integration Grant (CIG), it was furthermore concerned with building up infrastructure for research at the fellow’s new host institution in Scotland, UK. These two concerns, on the one hand, led to originally unplanned but very successful work on improved methods for WildClocks research. We established and summarised three publications several innovations for recording the birds’ biological rhythms in the field. The concerns also had the effect of adjusting the project goals to be locally more feasible. Adjustments involved an extension of study species to also include the locally more common Blue tit (Cyanistes caeruleus), in addition to the originally targeted but locally less common Great Tit (Parus major). Adjustments also involved a re-focus from variation in timing at a single site, forest habitat surrounding the University field station SCENE, to a comparative study of the birds at two sites representing different temporal habitats. This was done by contrasting the behaviour and physiology of the birds at SCENE with that of conspecifics at urban study sites in Glasgow. Thus, thanks to the CIG WildClocks grant we developed as a new study system the “Glasgow Gradient”, from which we have published several papers. We particularly focused on differences between urban and forest birds that are likely related to effects of artificial light at night (ALAN) which is known to disrupt circadian clocks. Working with these wild animals generated an originally unintended complexity of environmental conditions because the contrasting habitats differed in many ways, in addition to ALAN (see below). Finally, developments in the wider research community provided much more molecular background on the study species. Based on this we refocused from the original plan of genomic comparisons to a greater consideration of expression of genes. All this implied a widening of the original plans, which we see as a strength of flexibly adjusting the research agenda to promising and tractable conditions.

At the end of the first reporting period our tools, from telemetry and computational scripts to metabolomics, had largely been successfully developed. By then, we had established the foundations for a long-term study system of biological time-keeping across different temporal habitats (along the Glasgow gradient) and levels of biological organization (from behaviour to genes). In the second reporting period, we used this infrastructure and tools to better understand time-keeping, fitness and body condition of birds in anthropogenically modified environments. To this end we carried out observational and experimental studies of behaviour, physiology and gene expression of Great and Blue tits along Glasgow Gradient. Nonetheless, the second reporting period also presented us with new challenges: firstly, we found that a large proportion of the young birds we studied were acutely infected by avian malaria. This required consideration because malarian blood parasites show rhythmic development, which interacts with the host’s circadian rhythms. We are unaware of earlier studies that took account of avian malaria in the context of clocks, but saw it as both a necessity and opportunity to include it in our study. We strongly felt that consideration of natural infections, which are normally not incurred in captive animals, would greatly enrich studies of clocks in the real world. Fortunately, the host institute’s diverse research community offered strong collaborators who helped to extend the project to include a research line on avian malaria. Secondly, we found that the complexity of environmental conditions required consideration of a broader range of genes than could feasibly be analysed by PCR methods. Hence, we established newly for wild birds a plex assay that simultaneously assesses expression of multiple genes from small amounts of RNA. Inclusion of the two new elements enrich the scope of the project but also mean that our empirical work is still in full swing. Results are produced as I am writing, mostly as by two final-year PhD students.

Based on the four years of WildClocks research support our prediction that birds studied in urban areas, where their clocks are subjected to disruptive ALAN, are in worse body condition than in dark forest areas. The evidence includes several positive indicators of condition and health, such as reproductive success and nestling body mass, that are depressed in urban areas. In contrast, several negative indicators, including levels of the stress hormone corticosterone and of expression of inflammation genes, are increased in city birds. Some of these findings were recently published in a theme issue of effects of urbanization to wild species. However, singling out the link between these effects and biological clocks needs elaboration because our forest and city sites clearly differ in various aspects, not only ALAN. Therefore, while the findings are suggestive, they could also be due to other environmental factors that differ between city and forest. A primary candidate, on which we report in a publication, are differences in nestling food type and quality between the city and forest. To clarify the role of ALAN, as opposed to food and other factors, we applied small dim lights light to nestboxes at SCENE. Although we are still awaiting gene expression data, preliminary results suggest that the lights have indeed suppressed nestling growth. Whatever the causes of differences in body condition and reproductive success in urban birds may be, we see no indication that they take effect prior to hatching. Based on studies from 2015-2017, we found no evidence for differences between urban and forest birds relating to genetic background or to maternal hormones deposited to the eggs.

In addition to these extensive empirical studies, we also contributed a series of conceptual papers to the emerging field of Wild Clock studies. As explained above, this was necessary because the different fields on which the field is based are poorly integrated. Several of these studies were published in a highly visible, dedicated theme issue of Philosophical Transactions of the Royal Society entitled “Wild Clocks”, which was also edited by the Curie fellow and published in 2017. The theme issue is intended to become a milestone for research on wild clocks. The written work was paralleled by active dissemination of results and ideas at various scientific conferences and also on many occasions outside of academia.

Overall, we see WildClocks as a highly dynamic project that was flexibly adapted to local conditions and scientific developments, yielding a rich harvest of scientific findings. Clearly, much work remains to be done, but the developed infrastructure and methods, and the enthusiastic group in place will continue to delineate the role of clocks in the wild. Unfortunately, I, as the fellow, will locally in Scotland be only marginally involved in this future research. Following the UK referendum results (“Brexit”), I decided that the future of the lives of myself and family should lie within the EU. Therefore, I accepted a position in the Netherlands, but I will continue to work with colleagues in Glasgow. The young researcher employed by the CIG has secured a permanent position in Glasgow, and I will be a Visiting Professor to my current host institution, in addition to a professorship in the Netherlands. Thus, in the future the Wild Clocks project will continue in close collaboration and perhaps function as a bridge between post-Brexit Scotland and the Netherlands.