Gut health and immune function: the emerging role of gut microbiota in sustainable aquaculture

Lead Research Organisation: University of Aberdeen
Department Name: Inst of Biological and Environmental Sci

Abstract

Global salmon production is ~1.5 million tonnes, with the UK currently at ~170,000 tonnes, which is worth approximately £1 billion to the UK economy annually. The majority of UK salmon aquaculture is located in Scotland and it represents over 40% of all Scottish food exports. Salmon farming is a growing food industry sector and Scotland has an ambition to increase its production by 50% by 2020. A major bottleneck of this growth is the provision of high quality feed that does not adversely impact salmon health. Salmon are piscivorous and traditional salmon feeds rely on wild sources of fish protein and fish oil that can no longer meet the demand from the global aquaculture industry. To overcome this shortfall, plant proteins and vegetable oils are being used to replace the wild sourced fish in aquaculture feeds. There are a number of major problems and constraints associated with this replacement, including reduced nutrient digestibility, gut inflammation, gut microbial imbalance and impaired resistance to pathogens.

In this project, we will perform a series of experiments on Atlantic salmon and a zebrafish model fed with fish meal and plant-based diets to understand how these different diets shape the relationship between early intestinal development, immune function and gut microbiota. Zebrafish provide a unique model to study such a complex relationship because they are translucent and have established many advanced experimental techniques, including generation of germ-free larvae, live imaging of larvae colonised with fluorescent-tagged bacteria and generation of fish with fluorescent protein labelled inflammatory cells. None of these techniques are currently available for salmon or other farmed fish.

By performing salmon feeding trial, we will establish how plant-based diets affect gut health (changes in gut morphology and gene expression profile) and how they change the abundance and diversity of gut microbiota. The diversity of gut microbiota will be determined by the state-of-the-art next-generation sequencing of 16S rRNA. Crucial to intestinal health is the establishment of a well-balanced gut microbiota community, which frequently happens at the time of first feeding. At this stage, the intestinal immune system is not mature and allows colonisation of the gut by symbiotic bacterial species. We will use zebrafish to study these early life events, with the key goal to test whether manipulation of gut microbiota improves the oral tolerance to sustainable aquaculture diets with high inclusion of plant proteins and vegetable oils. Thus, the microbiota swap between zebrafish larvae fed with fish- or plant-based diets may open up new avenues for investigating pathways to improved health and immune function in farmed fish. Finally, both salmon on different dietary regimes (and associated changes in gut microbiota) and zebrafish with manipulated gut microbiota will be exposed to a bacterial pathogen to determine whether the capacity of the fish to fight the infection depends on prior intestinal condition and microbiota community. The important part of the project will be development of molecular diagnostic tools for fish gut microbiota, enabling rapid screening for beneficial and detrimental sets of bacteria in fish guts.

The main outputs of the project include:
1) Identification of key microbiota species associated with gut health and intestinal dysfunction in farmed fish (salmon) and a model species (zebrafish).
2) Evaluation of the capacity of gut microbiota to modify gut function and applicability of gut microbiota transfer to improve oral tolerance of farmed fish to plant-based diets.
3) Development of assays for detection of microbiota species, which can be used to create diagnostic tests for gut health.

Technical Summary

To enable continued expansion of the salmon aquaculture industry, feeds based on plant ingredients need to replace traditional diets sourced from wild-caught and over-exploited fisheries. However, high inclusion of plant proteins and vegetable oils are known to contribute to gut dysfunction and induce gut inflammation (enteritis). In this project, we will perform a series of experiments on Atlantic salmon and a zebrafish model fed with fish meal and plant-based diets to understand how these different diets shape the relationship between early intestinal development, immune function and gut microbiota. The ultimate aim of the project is to test whether manipulation of gut microbiota may improve oral tolerance of farmed fish to plant-based diets, and thus, provide a completely novel approach to nutritional programming in sustained aquaculture. The salmon feeding trial will identify bacterial species (16S rRNA next-generation sequencing) that become more prevalent under different dietary regimes and evaluate associated changes in gut health (morphology and gene expression profiling). Utilising gnotobiotic (Gn) zebrafish with fluorescent protein (FP) labelled inflammatory cells (neutrophil/macrophage-FP fish) and fluorescent-tagged gut microbiota will enable to perform a proof-of-concept experiment, during which Gn larvae will be inoculated with bacteria derived from intestinal content of conventionally-raised zebrafish (fed with fish meal or plant-based diet) and then exposed to these diets in later life. Both salmon on different dietary regimes (and associated changes in gut microbiota) and zebrafish with manipulated gut microbiota will be exposed to a bacterial pathogen to determine whether the capacity of the fish to fight the infection depends on prior intestinal condition and microbiota community. Molecular assays, generated within the project, for salmon and zebrafish will be of scientific and commercial interest.

Planned Impact

Our main goal is to elucidate the relationship between gut health, diet and microbiota in fish. We will take a dual approach to address our objectives, using both Atlantic salmon and a zebrafish model, in which the availability of more sophisticated tools can greatly enhance the early life immunological aspects of the work. Our key aspiration is to increase the tolerance of plant-derived nutrients in farmed fish, by identifying indicator bacterial species for gut health and also by the potential to induce oral tolerance in early life stages of fish. The key target audience is both academics and industrial stakeholders working in a variety of fields, including fish nutrition and health, fish immunology and gut microbiology. The project has an industrial partner BioMar Ltd (a multinational fish feed manufacturer), with the project scope fitting directly into their research and development plans. As the availability of fish meal and fish oil decreases, the requirement for alternative nutrient sources (such as plant proteins and vegetable oils) is increasing year on year. Currently, there is no in-depth information on how these plant-derived diets will modify gut microbiota, nor is there significant knowledge of the establishment of the microbiota and parallel development of the immune system.

The project will have joint meetings twice per year and a regular part of the agenda will be to review the project impact and dissemination. Prof. Simon Davies (co-I, PU) will take on the role of lead in terms of impact to ensure that the impact of the research is realised. Prof. Davies is the editor of International Aquafeed, a widely read magazine bridging the gap between industry and academia and has many years of experience with interacting with both industrial partners and academics. We will aim to publish annual articles in high impact factor journals relating to microbiology and health, such as The ISME Journal, British Journal of Nutrition and BMC Genomics. In addition, reports for Fish Farmer Magazine and International Aquafeed will highlight the results to the industry. To reach additional audiences, our industrial partner is a sponsor of several major international meetings organised by the World and European Aquaculture Societies. Prof. Martin is a forum member for the aquaculture theme within The Marine Alliance for Science and Technology for Scotland (MASTS) and was also a member of the Scottish Government Ministerial Group on Sustainable Aquaculture. These are both avenues through which the impact of our project targets key governmental, industrial and strategic plans for aquaculture research. The use of zebrafish as a model for fish health and nutrition is in its infancy, but Drs Lawrence and McGonnell will transfer our applied work to the zebrafish community by dissemination of the results in the scientific journals and during the local and international conferences. We will develop a diagnostic platform to monitor salmon intestinal health that will directly contribute towards development of new diets and reduce the time scale for testing of new diet formulations. The outputs of this project will also help to meet the 3Rs by improving the screening approaches of new dietary ingredients.
 
Description The impact of different diets on Atlantic salmon have had minimal impact on the intestinal morphology and this is confirmed by examining gene expression in the intestine by both microarray and RNAseq. Examination of microbiome in the distal intestine of the Atlantic salmon indicted some subtle changes in Operational Taxonomic Units, further study may help link these changes to function.
A key outcome from the project was the recognition for a greater interaction of the academic community in the UK working on microbiota in aquaculture systems. as such a UK wide workshop on microbiota in aquaculture was held by the ARCH UK aquaculture hub in Aberdeen during November 2018. This had approx 70 delegates and led to new networking opportunities for many ECRs.
Exploitation Route Once published further knowledge on the impact of dietary changes on intestinal health and microbiota may be used to help design new diets for aquaculture.
Sectors Agriculture

Food and Drink

Environment

 
Description Investigating the potential of nutritional programming to improve the utilisation of sustainable feeds in aquaculture
Amount £673,766 (GBP)
Funding ID BB/R018812/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2022
 
Description Novel Omega-3 Sources in Feeds and Impacts on Salmon Health
Amount £726,052 (GBP)
Funding ID BB/S005919/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 12/2024
 
Description ROBUST-SMOLT Impact of early life history in freshwater Recirculation Aquaculture Systems on A. salmon robustness and susceptibility to disease at sea
Amount £195,126 (GBP)
Funding ID BB/S004270/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2022
 
Title Microarray for Atlantic salmon 
Description We have used microarray for gene expression in Atlantic salmon to understand the gen expression responses between different life history stages. We have fully reannaotated the microarray to make down stream data analysis much better. To do this we associated each gene feature on the microarray with a human protein identifier, allowing programs such as DAVID and KEGG analysis to be used. 
Type Of Material Biological samples 
Year Produced 2011 
Provided To Others? Yes  
Impact papers in preparation 
 
Title microbiome analysis 
Description Determination of the microbiota in Atlantic salmon. This has used 16S sequencing by Illumia to determine bacterial species in salmon. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Initial work still under analysis 
 
Description Dr Aleksei Krasnov 
Organisation NOFIMA Ås
Country Norway 
Sector Private 
PI Contribution Development of tools for assessment of the immune competence of Atlantic salmon smolts and growers (Forskerprosjekt - HAVBRUK2)
Collaborator Contribution Development of tools for assessment of the immune competence of Atlantic salmon smolts and growers (Forskerprosjekt - HAVBRUK2
Impact Project just started no outputs yet
Start Year 2017
 
Description Epigenetic management of stress and disease resistance in Atlantic salmon 
Organisation Swansea University
Country United Kingdom 
Sector Academic/University 
PI Contribution RNAseq and analysis of juvenile salmon
Collaborator Contribution RNAseq and analysis of juvenile salmon
Impact No Outputs published as yet
Start Year 2015
 
Description 2nd International Conference of Fish & Shellfish Immunology, Portland, Maine, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral Conference presenation

GUT HEALTH AND INTEGRITY IN ATLANTIC SALMON FED PLANT PROTEIN DIETS
Elzbieta Król, Alex Douglas, Christopher J Secombes and Samuel A. M. Martin
2nd International Conference of Fish & Shellfish Immunology, Portland, Maine, USA. 26-30 June 2016
Year(s) Of Engagement Activity 2016
 
Description European Aquaculture Society meeting 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact BBSRC grant BB/M026604/1 Gut health and immune function: the emerging role of gut microbiota in sustainable aquaculture.
Impact plan to host a session at the Aqua 2016 conference on "Recent Advances in Digestive Physiology: The Gut, its Health and Molecular Nutrition" 22nd September 2016.
Year(s) Of Engagement Activity 2016
 
Description European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral Conference presentation
E. Antonopoulou, K. A. Kormas, S. Chatzifotis, G. Piccolo, L. Gasco, SAM. Martin and E. Mente
PROTEOMIC ANALYSIS IN SEA BREAM S. aurata, SEA BASS D. labrax AND TROUT O. mykiss FED WITH DIETS CONTAINING INSECT MEALS
European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016
Year(s) Of Engagement Activity 2016
 
Description European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presenation
Rodiles, A., Tinsley, J., Martin, S.A.M., and Merrifield, D. L.
EFFECT OF MARINE AND PLANT-BASED DIETS ON THE GUT MICROBIOME OF THE ATLANTIC SALMON (Salmo salar)
European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016
Year(s) Of Engagement Activity 2016
 
Description European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016 Krol et al.,2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral Conference presenation
E. Król, A. Douglas, D. R. Tocher, V. O. Crampton, C. J. Secombes and S. A. M. Martin
PLANT PROTEINS IN AQUACULTURE FEEDS: NEW INSIGHTS FROM THE GUT TRANSCRIPTOME OF ATLANTIC SALMON
European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016
Year(s) Of Engagement Activity 2016
 
Description European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016 mccarthy et al., 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral conference presentation

Ú. McCarthy, R. Pettinello, L. Feehan, Y.M. Ho, P. White
EXPERIMENTAL TRANSMISSION OF SEGMENTED FILAMENTOUS BACTERIA (SFB) IN RAINBOW TROUT (Oncorhynchus mykiss, WALBAUM)
European Aquaculture Society, Aquaculture Europe, 20-23rd September 2016
Year(s) Of Engagement Activity 2016
 
Description ICAR- Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700120, India 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Sam Martin. Invited International seminar INDIA
Future nutritional challenges, in Atlantic salmon with focus on Scottish Aquaculture. Invited seminar. ICAR- Central Inland Fisheries Research Institute, Barrackpore, Kolkata 700120, India. 23rd Febuary 2016
Year(s) Of Engagement Activity 2016
 
Description Invited Seminar, Roslin Institute, University of Edinburgh 14th March 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Sam Martin
Atlantic Salmon; Future nutritional challenges.
Invited Seminar, Roslin Institute, University of Edinburgh 14th March 2016
Year(s) Of Engagement Activity 2016
 
Description Merrifield, D. L. (2019) Probiotics in aquaculture: current research and future focus. Biomin Aquadays 2019, August 27th-29th, Vienna, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Industrial focussed research presenation on use on probiotics in fish feed. Special reference to microbiota.
Year(s) Of Engagement Activity 2019
 
Description Nutrition and Feeding, Sun Valley, Idaho, USA 5-10 June 2016 Dehler et al., 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation at conference
Carola E. Dehler1, Christopher J. Secombes1, Samuel A.M. Martin1
Microbiota and gut health in Atlantic salmon (Salmo salar). International Symposium on Fish Nutrition and Feeding, Sun Valley, Idaho, USA 5-10 June 2016
Year(s) Of Engagement Activity 2016
 
Description Sam Martin: Aquaculture: Future Nutritional Challenges. British council and Gulf Science and Innovation. Agritech , Aquaculture and Food Safety Forum, Muscat, Oman, 5th March 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact International workshop between British Embassy, Oman and the Gulf Stes Science program.
Year(s) Of Engagement Activity 2018