FUTUREFISH: The role of circadian rhythms, immunity and infection in enhancing aquaculture

There is a rapidly increasing global demand for fish, yet stagnation and collapse of traditional capture fisheries, therefore aquaculture (fish farming) must be expanded and intensified to meet demand. Currently, the principal barrier to aquaculture development is disease; causing devastating economic losses and increasing reliance on drug interventions. Indoor fish farming provides hope for intensification of aquaculture; finely controlling environmental conditions to maximise fish production without encroaching on natural aquatic habitats. However, while manipulated light regimes (extended day lengths or even continuous light) are often used in indoor aquaculture to improve rearing, it is unknown how such approaches contribute to disease issues.

Across all forms of life; from microbes to humans, organisms exhibit daily cycles in biological processes such as behaviour and metabolism, known as circadian rhythms. Circadian rhythms are typically controlled by expression patterns (turning on and off) of "clock" genes; a system often referred to as the molecular body clock. Molecular clocks can be altered or "entrained" to light conditions. Body clocks and circadian rhythms are important to immune system functioning; levels of immune cells and the ability to fight infection varies by time of day in mammals. Moreover, disruption of circadian rhythms (e.g. shift-work, jet-lag) can increase susceptibility to or severity of disease in humans. Therefore, the characterisation of fish circadian rhythms of immunity and the examination of how light regimes affect fish disease resistance is vital to future sustainable improvement of aquaculture methods. In addition, although it is well known that parasites too exhibit daily rhythms, we do not yet know the nature of their molecular body clocks. Therefore, to fully understand the daily interplay between hosts and their pathogens, be it fish or other animals, we must also consider the control of rhythmicity in parasites.

Working at Cardiff University, bringing together world leaders in fish parasitology, invertebrate genetics, and microbial communities, and in collaboration with experts in molecular clocks at Aberystwyth University, I will address these key knowledge gaps in the circadian biology of fish-parasite interactions. I will measure the impact of three economically-important parasites (Argulus; freshwater louse, Gryodactylus; skin worm, Saprolegnia; water mould) on rainbow trout circadian rhythms of clock and immune gene expression, and determine how light regimes alter the susceptibility of trout to infection. It is predicted extended and/or constant photoperiods increase susceptibility to disease. In addition, I will quantify the circadian rhythms of gene expression in Gyrodactylus and couple the findings with measures of parasite activity and infection ability. I hypothesise rhythmicity of key genes related to parasite infection ability, coupled with daily variations in host immune levels, drive circadian rhythms in parasite survival and behaviour on-host. Finally, I will determine whether the microbiome (bacteria community) of trout skin exhibits circadian rhythmicity and examine how perturbation, via antibiotic treatment, impacts the skin's microbial composition and subsequent susceptibility to parasites. It is predicted reduced variation in beneficial skin microbes due to antibiotics will increase susceptibility to parasites.

Harnessing the power of optimal circadian rhythms can improve health and reduce antimicrobial usage in aquaculture. Taken together, the results of this fellowship will provide important new insights to inform fish farm management practices and fundamentally advance the understanding of daily interactions between parasites and their hosts.

Disease outbreaks currently hinder the economic growth of intensive aquaculture, and increasingly are the key factor leading to closure of fisheries. For indoor fish farms, photoperiod manipulation is used to improve growth and maturation rates. However, how such practices contribute to current disease problems is unknown. Circadian rhythms are ubiquitous to life and central to this rhythmicity is the expression of "clock genes", which regulate rhythms in key biological processes including immune functions. Whilst the intricate links between clock genes and immunity are recognised for mammals, the molecular mechanisms underlying parasite rhythmicity is poorly understood. Therefore, an integrated chronobiological approach to host-pathogen interactions is required to enhance fish production yield.

Utilising a multi-model fish-parasite system (Oncorhynchus mykiss-Gyrodactylus, Argulus, Saprolegnia), I will apply behavioural, experimental, qPCR techniques to assess how interactions between light regimes and parasite infections alter activity, clock and immune gene expression, and disease susceptibility. I will employ high-throughput RNA sequencing to reveal rhythmicity of the Gyrodactylus transcriptome in relation to circadian patterns of parasite activity and transmission potential. Finally, using 16S rRNA gene profiling, I will assess the rhythmicity of commensal microbiota of O. mykiss skin and examine how Gyrodactylus infection and antibiotic treatments alters skin microbiomes. For the first time, this will 1) examine the impact of ectoparasites on host circadian rhythms, 2) test the effect of photoperiod manipulation on fish disease susceptibility, and 3) uncover the molecular control of circadian rhythms in parasites. By incorporating rhythmicity of host, commensal microbiota, and parasite, into the first skin interactome, this project will provide for a step change in development of holistic chronobiological approaches to animal health and disease mitigation.

Globally, aquaculture is the fastest-growing food sector due to the rapidly increasing demand for fish protein and diminishing traditional capture fisheries. Disease is the principal barrier to economic development and sustainable intensification of aquaculture; accounting for losses of ~US$6 billion per year worldwide and driving increasing reliance on drug interventions. Indoor aquaculture promises the benefits of environmental manipulation to maximize production, without the detrimental impacts on natural aquatic ecosystems. Extended and even continuous light regimes are often implemented to increase growth and alter maturation rates of fish. However, I propose, given our growing understanding of the intricate links between circadian rhythms, molecular body clocks, and health in mammals, that such practices may substantially contribute to current aquaculture disease problems. Moreover, we lack a fundamental understanding of the molecular control circadian rhythmicity of parasites, requisite to the advancement of chronotherapeutic strategies for animal and human health. Furthermore, host commensal microbiota and their interaction with pathogens, plus potential dysbioses due to drug treatments must be considered for a holistic approach to health. Thus, furthering our knowledge of the circadian interactions of fish, their parasites and microbiota will provide immediate economic and societal beneficiaries in animal health and food security. In addition, providing grounding for novel chronobiological parasite control and microbiome manipulation measures as viable alternatives to traditional pharmaceuticals, this project has longer-term prospects for commercial outcome.

Animal health & food security: Disease and a lack of viable control measures continues to be a central issue to sustainable aquaculture development at a national and international level. Findings from this project, particularly evidence of altered disease susceptibility under different light regimes, will be immediately applicable to aquaculture facilities managers. To help mitigate infection risks by applying a healthy circadian environment for fish stocks, this will ultimately increase aquaculture economic productivity and reduce use of drug treatments. A commercial aquaculture systems design company (Aqua-Ecosystems) has already expressed interest to incorporate findings into their current recommendations for clients.

Policy & commercialisation: In addition to direct dissemination to the aquaculture trade, this data should also benefit the instigation of new codes of best practice for aquaculture regulators such as the Fish Health Inspectorate at Cefas, and DEFRA. Furthermore, the impacts of circadian light environment on animal health will be more broadly applicable to terrestrial livestock management such as indoor poultry rearing practices. As the work proposed may provide routes towards novel fish therapeutics, such as time-of-day targeted treatments and bioaugmentation of microbiota, the longer-term commercial impact of this fellowship has been recognized and IP potential will be explored with the support of Cardiff Research & Innovation throughout the project (see Pathways to Impact).

Public engagement & impact: By identifying circadian variation in parasite infectivity and their molecular body clocks, this fellowship has the potential to substantially contribute towards our understanding of both veterinary- and medically-important parasitic infection dynamics. The public is fascinated with parasites and the ways in which they can affect our everyday lives, as seen by interest in the high-profile TV series 'Monsters Inside Me' (Discovery Channel) and 'Embarrassing Bodies' (Channel 4). The potential outcomes of the proposed research will impact on the general public by raising awareness of how important light periods and body clocks are to fight parasitic infections (see Pathways to Impact).