A microbial basis for Atlantic Salmon energetics

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Production of the global salmonid aquaculture industry now exceeds 2.4 megatons per annum. Major European producers expect to expand their outputs between 30-50% over the next five years. Ambitions for expansion on this scale create major concerns around fish welfare, ecological impact and the sustainability of salmon feed components.
Nutrition lies at the heart of the issue. In the wild, Atlantic salmon are specialized carnivores. In aquaculture, in a move away from the unsustainable use of wild fish protein and oil, proteins of plant origin now constitute the majority (>60%) of their diet. Associated digestive abnormalities are common. In addition, plant-based diets may affect the rate at which nutrients are absorbed and the associated growth rate of salmon, which determines how quickly they grow in marine cages. Rapid marine growth is desirable since it permits more extensive fallowing of coastal aquaculture sites, which reduces the impact of farmed fish on the marine environment (pathogen transfer, nutrient pollution). Finally, while wild fish protein can be replaced by plant protein in salmon diets, oils cannot. Key omega-3 fatty acids must be sourced from the marine environment; a significant burden on wild fisheries. Ensuring the efficient assimilation of fatty acid components from salmonid diets is of paramount importance to safeguard the sustainable exploitation of marine resources.
Salmon energetic phenotypes are composites of several interlinked traits: metabolic rate, body fat content, growth, energy harvest from food, energy economy in times of starvation. These traits underpin concerns around salmon nutrition. Significant energetic variation exists in both wild and farmed salmon with multiple possible drivers - both genetic and environmental. Importantly a wealth of new data indicates a role for intestinal microbiota - the bacteria that live in the guts of all vertebrates - in determining host energy metabolism.
Understanding how gut bacteria influence Atlantic salmon energetics is thus fundamental to understanding their role in nutrition. This is the principal aim of this project. To achieve this we will establish the influence of gut bacteria on the energetics of salmon living in salt and freshwater, in both wild and aquaculture settings. First, in a unique experimental river system established in Burrishoole, Mayo (Marine Institute/University College Cork, ROI) we will track introduced juvenile salmon through their freshwater lifecycle, measuring both metabolic and gut microbiome variation. This 'wild' cohort will include wild and released farmed fish to establish whether differences between gut microbiota contribute to the poor performance of farmed juveniles in the wild. Secondly, we will undertake parallel freshwater experiments in simulated aquaculture conditions in the laboratory at the University of Glasgow (UoG),UK. Finally, in association with Marine Harvest, we will carry out corresponding experiments on saltwater phase pre-adult salmon in Norway.
Alongside experiments with live fish, we propose to harness bioengineering expertise at the School of Engineering, UoG and biotechnological expertise via industry partner Alltech to build an artificial salmon gut system. Via the transfer and maintenance of gut bacteria from metabolically different fish from farmed and wild settings into our gut model we aim to establish how bacterial fermentation underpins differences in energy harvest from feed. Once these 'artificial' bacterial communities are established, a final exploratory phase of the project will involve their transplantation into laboratory reared juvenile salmon to evaluate their potential impact on host metabolism.
Understanding how salmon gut bacteria change energetic phenotypes will open new avenues to improve fish health, nutrition and productivity. A model Atlantic salmon gut in a world class UK bio-engineering laboratory puts in place an invaluable tool for salmon aquaculture in the UK

Technical Summary

Atlantic salmon are anadromous salmonids of major commercial, cultural and recreational importance, in the UK, Ireland and worldwide. Metabolism, feed conversion efficiency and growth lie at the core of salmonid aquaculture productivity and its ecological impact and sustainability. The role of gut microbiota in driving energy metabolism in vertebrates is increasingly clear, opening up new avenues to fine-tune salmon metabolism and growth.

The aim of this project is to establish the microbial basis for different energetic phenotypes in Atlantic salmon. In doing so, we will establish the role of microbiota in influencing host performance and energy economy. The broad project objectives are as follows:

1)Determine the links between energetic phenotype and microbiota in the natural environment, in order to explore the full variation in both sets of traits.
2)Examine the same links in fish of different life stages but in laboratory/farm settings where food is continually available and where nutritional intake can be standardised and measured.
3)Establish the role of microbiota in driving maladaptive energetic phenotypes in farm escapes and hatchery reared fish deliberately released into the wild.
4)Establish a synthetic salmon intestinal microbiome system through which to validate the energetic profiles we find in farmed and wild fish.
5)Undertake pilot microbiome transplantation to establish the causal role of microbiota in determining the phenotype of the host.

The project brings together: Irish and UK expertise in fish biology, population genetics, microbial ecology; a world class UK bio-engineering laboratory; and major industrial partners (Marine Harvest, Alltech) to open new avenues to improve fish health, nutrition and productivity. The establishment of a model Atlantic salmon gut puts in place an invaluable tool for salmon aquaculture that will boost the UK's aquaculture research capacity in the lifetime of the grant and into the future.

Planned Impact

Production of the global salmonid aquaculture industry now exceeds 2.4 megatons per annum. Major European producers (e.g. Norway, Scotland, Ireland) expect to expand their outputs between 30-50% over the next five years. Ambitions for expansion on this scale create major concerns around fish welfare, ecological impact and the sustainability of salmon feed components.

Nutrition lies at the core of many of the underlying concerns. Thus, addressing knowledge gaps around salmon nutrition is crucial to ensuring the sustainable expansion of the aquaculture industry. This project addresses knowledge gaps by 1) Establishing the role of gut microbiota in driving nutritional energy harvest, nutrient assimilation, metabolic rate and growth in farmed and wild Atlantic salmon. 2) Establishing and validating an artificial salmon gut system to test novel feed additives aimed at improving sustainable aquaculture production and improving fish welfare. Impacts on fish welfare include not only improved feed formulations, but also a significant potential reduction in the number of in vivo trials necessary for feed development.

The principal beneficiaries of this research are the UK and Irish economies. Combined, their aquaculture industries support >10,000 jobs directly, and many more in service economies indirectly - especially in often rural and remote communities with otherwise limited access to employment. The proposed project promises to improve the efficiency of salmon growth and nutrition. In doing so it will positively benefit the competitiveness of the UK and Irish salmonid aquaculture industries, paving the way for sustainable growth and expansion of a valuable export economy.

The aquaculture industry stands to benefit significantly from this research. Recognition of this fact in the first instance is the cerca £300,000 in-kind and cash contribution committed by industrial project partners. In a recent industry-wide poll of research interest areas at a recent Scottish Auaculture Innovation Centre workshop (http://scottishaquaculture.com/events/sustainable-aquafeed-workshop), themes linked to salmon gut microbiota, novel feeds, and sustainable use of feeds (including DHA and EPA from fish oil) were identified as the top three areas of concern and interest. Our research findings with respect to the microbial drivers of Atlantic Salmon energetics, as well as the artificial gut tool we will develop - with potential for testing novel feeds, feed additives and microbial therapies - is strongly aligned with industry needs.

Ultimately, via the improved understanding of the role of microbiota in salmon energetics nutrition that this project offers, there is potential to improve the efficiency of salmon aquaculture and concomitantly: 1) reduce the impact of intensifying salmonid aquaculture on coastal ecosystems as well as 2) reduce the reliance of salmonid aquaculture on wild capture fisheries.
 
Description A microbial basis for Atlantic salmon energetics

Work Package Leader: Dr Martin Llewellyn & Dr Philip McGinnity

Atlantic salmon (Salmo salar) are anadromous salmonids of major commercial, cultural and recreational importance in the UK, Ireland and worldwide. Metabolism, feed conversion efficiency and growth lie at the core of salmonid aquaculture productivity and its ecological impact and sustainability. The role of gut microbiota in driving energy metabolism in vertebrates is increasingly clear, opening up new avenues to fine-tune salmon metabolism and growth. This multidisciplinary project brings together world class UK and Irish fish biologists, population geneticists, microbiologists, bio-informaticians, engineers and major industry partners to determine for the first time the role of salmon gut microbiota in defining host energetics, so paving the way for more sustainable salmon farming.

OBJECTIVES:

Objective 1: To establish the link between salmon energetic phenotype and microbiome diversity a) in a population of wild juvenile salmon that will express natural variation in both traits; and in laboratory-reared fish in simulated aquaculture conditions at the b) freshwater and c) marine life stages.

Objective 2: To establish the role for intestinal microbiota in driving maladaptive energetic phenotypes in farmed salmon escaped or released into the wild using: a) domesticated farmed fish and b) captive-bred wild fish.

Objective 3: To establish the basis for microbial energetic differences using ex-vivo and transplantation approaches via a) development of an artificial salmon microbiome system to validate microbial energetic profiles we find in fresh and saltwater settings, and b) transplantation of ex-vivo microbiota into salmon, sequence-based evaluation of transplantation success and energetic phenotype follow-up.


Progress & Results
Details of progress and results to date in relation to the WP Tasks, Deliverables and Milestones.
Recruitment to the Irish element of the project is on schedule with appointment on 1 December 2016 of Dr Jamie Coughlan (UCC) as Post-Doctoral Researcher (PDR) and Dr Fintan Egan (UCC) as Research Assistant. The project at Glasgow University does not commence until 1 April 2017. The project is on schedule

Methodology and research activities (tasks) to address Objectives 1 & 2, 3.

(1) Experimental populations of wild-origin (WO), farm-origin (FO) and ranched-origin (RO) salmon (15 families per population) will be established by hand-fertilising stripped eggs in the spawning season of year 1.

• 2018 Report - In addition to the experimental population established last year (2016/17 cohort) consisting of the progeny of pure wild origin, farm origin and ranch origin, we have produced a new population (2017/18) made up of the progeny of farm and wild salmon including their hybrid progeny. These fish provide excellent extra study opportunities, particularly the hybrids, both under freshwater hatchery conditions and at the new marine out-growing site at Connemara. (2) The WO population will be offspring of wild fish collected in the Burrishoole river system, Ireland.


• Report 2019: The 2017/18 cohort experimental population has been successfully established under natural (wild) conditions in the Rough River, under hatchery conditions at the Newport salmon hatchery and under marine captive conditions at the Marine Institute's sea farm at Bertreach Bui Co. Galway. Ova were also transferred to the University of Glasgow (UoG) to establish a population in aquaria in March 2018
• Report 2020: The 2017/18 cohort was extensively sampled in the rough river section in summer to collect microbial communities from intestines. No further cohorts fish were seeded into the river, as per the plan. Final sampling of fish is occurring to coincide with the smolt run in spring 2020


(2) The WO population will be offspring of wild fish collected in the Burrishoole river system, Ireland.
• 2018 Report - Completed as planned for 2017 with an additional wild origin group established in January 2018.

• Report 2019: Both experimental cohorts have been monitored continuously at the Rough River trapping facility since their introduction into the experimental section of the river. The WO population was also established at the UoG with experimental work ongoing on 2016/17 and 2017/18 cohorts.

• Report 2020: the 2017/18 cohort will go out to sea as smolts this year, finishing the experiment.


(3) The FO parental fish, which are of Norwegian provenance, will be provided by Marine Harvest and represent the most commonly grown farm strain in Ireland.
• 2018 Report - Completed as proposed for 2017 with an additional farm origin group established in January 2018.

• Report 2019: As above for WO fish

• Report 2020: As above for WO fish



(4) The RO fish will be hatchery-reared juveniles from a ranched population (i.e. where fish are reared in the hatchery to the smolt stage then released into the wild, with returning adults forming the next brood stock).
• 2018 Report - Completed as described for 2017 with an additional ranch origin group which was established in January 2018.

• Report 2019 - Completed.

(5) Fertilisations will be conducted over a brief (3 day) period and samples of eggs weighed to facilitate comparison of subsequent growth rates. Fin clips from all adults will be retained for subsequent parentage assignment of offspring.
• 2018 Report - Tissue samples were collected from the parents to facilitate later parentage assignment of juveniles from the 2016/17 experimental population cohort. Biometric data was collected from the parents and the egg offspring. Similarly, tissue samples and biometric data have been collected for the 2017/18 cohort. A full genetic baseline of the parental fish used in the 2016/17 cohort has been completed to facilitate parentage of fish introduced into the river using 20 microsatellite markers.

• Report 2018: Complete



(6) Eggs from all three experimental populations (WO, FO, RO) will be maintained as family units within the hatchery facility at Furnace on the Burrishoole river system until needed for particular Activities as follows:

• 2018 Report - The experimental families are being managed in the Newport hatchery facility as described. The experimental population consists of 1,000 individuals of each of farm, wild and ranch parental group. The experimental population is growing well with the expectation fish will smoltify ready for introduction into sea cages in the spring of 2018. Depending on survival rates we will set up a new hatchery based experimental population in the spring of 2018.

• Report 2019: A new experimental population was established in 2018 and has been successfully on-grown. These fish will be introduced as smolts into the sea farm in spring 2019.

• Report 2020: Completed

(7) Eyed eggs from individual WO families (n = 2000 eggs from each of 15 families) will be counted, mixed and introduced into the experimental Srahrevagh River using artificial nests in early to mid-February.
• This task is on schedule. However we now plan to introduce the experimental population as swim up fry rather than eggs.

• 2018 Report - We introduced the experimental population into the river as swim up fry rather than eggs. The experimental population consisted of 8,213 individuals from 14 wild families and was introduced into the experimental river on 10 April 2017 and 19 April 2017. These were also 10,026 farm progeny released on 6 April 11,270 ranch progeny released on 7 April 2017. The 2017/18 cohort consists of 12 wild, 24 hybrid and 14 farm families and these will be put into the experimental river in April 2018, again as swim up fry.

• Report 2019: We introduced the experimental population from the 2017/18 cohort into the river as swim up fry (n=41,045). The introduction consisted of four groups: pure wild n=9,683; pure farm n=11,253; farm female x wild male hybrids n=11,815; wild female x farm male hybrids n= 8,294. The 2017/18 cohort consists of 12 wild, 24 hybrid and 14 farm families.
• Report 2020: The 2017/18 cohort was extensively sampled in the rough river section in summer to collect microbial communities from intestines. No further cohorts fish were seeded into the river, as per the plan. Final sampling of fish is occurring to coincide with the smolt run in spring 2020


(8) PIT-tagged hatchery-reared RO parr will also be introduced in late summer of the same year.
• Task scheduled for July 2017. Eggs will be ongrown in the hatchery until the summer parr stage of development.

• 2018 Report - The RO eggs were on-grown in the hatchery until the summer/autumn parr stage of development. A total of 291 RO 0+ pre-tagged, measured, and profiled for microbiota community hatchery reared parr were introduced (geolocations data collected for release points) into the Srahrevagh river experimental section over a two week period commencing the first week of October 2017.

• Report 2019: Of the 291 RO autumn released parr 160 were captured in the Rough River downstream trap of these 84 were smolts providing an autumn parr to smolt survival of 29%. A further 30 stock ranched smolts were captured at the Furnace main river traps. In addition there 62 observations stocked ranch parr during the electrofishing surveys. There were 30 gut samples acquired for microbiome analyses

• Report 2020: Complete
(9) In early autumn, 500 juvenile fish (approx.) will be collected in the experimental river by electrofishing, fin-clipped for retrospective DNA-based parentage and population assignment (following [4]), marked with PIT tags, and measured and weighed to determine growth rate (using family egg size as a covariate).
• Task scheduled for August/September 2017.

• 2018 Report - This task was undertaken in September and October 2017. The sampling was delayed due to high river levels following a series of autumn storm events. 99 0+ salmon were of a sufficient size to PIT tag. Measures of size at age were recorded to facilitate growth rate trajectories. The provenance of all the fish sampled were assigned to their parent population using the genetic baseline. The results of the genetic analysis suggest the groups of experimental fish are in similar proportions to the numbers stocked.

• Report 2019: The experiment was repeated in 2018 and is now complete for 2017 and 2018. 501 1+ fish from the 2016/17 cohort were captured of which 336 were PIT tagged

• Report 2020: Sampling has continued in 2019 with several large tagging and measuring campaigns (>1500 fish tagged)

(10) Using an on-site respirometry system, the SMR and AS of each fish will be non-lethally tested under standard conditions
• Seasonal metabolic status measures will commence September/October 2017. These will not be undertaken on-site but in newly developed facility at Furnace.

• 2018 Report - Seasonal metabolic status measures have still to commence on a routine basis. A new freshwater respirometry system (Loligo System, Denmark) has been established at the Newport facility. We are currently carrying out calibration exercises to optimise the system. Standard metabolic rate (SMR) measures have been undertaken for approximately 20 hatchery fish in order to optimise the system. The system when fully operational will be able to process fish from the size range of 8g to 100g. We are also currently exploring respirometry for smaller fish down to a gram in weight. We plan the establishment of a saltwater respirometry system also in Newport and to be operational in early spring.

• Report 2019: We have now fully installed and validated these systems on site. To improve sample throughput, we are also have been able to use ventilation rate (VR) as a proxy for inferring SMR and AS in a wider sample of individuals. Dr Chloe Heys, alongside Patrick Schaal and other UCC personnel are undertaking periodic sampling to assess seasonal fluctuations in metabolic state.

• Report 2020: Seasonal respirometry measurements have continued throughout 2019/202 including direct and indirect oxygen consumption measures.

(11) Whole body adiposity ratios will be determined non-lethally via a pressure system
• Seasonal assessments will commence autumn 2017

• 2018 Report - We are exploring the possibility of manufacturing a pressure apparatus approach to facilitate non-lethal adiposity measures. In meantime we will commence shortly the more conventional fatty acid profiling approach. We have been fortunate to secure an arrangement with the Teagasc Food Research Centre at Moorepark, Fermoy, to undertake this analysis. In mid-September six individuals from each of the experimental groups were sampled for fatty acid profiling. A further ten fish were also sampled from each group in mid-November. An additional 10 fish from each of the experimental groups were sampled in mid-January.

• Report 2019: For the experimental populations in the hatchery we are deploying a recently purchased fat meter (Distell FM902) to assess adiposity non-lethally. In fish lethally sampled for other purposes (e.g. microbiome) - fat measurements were assessed as a function of total body water content (using wet:dry weight ratios)

• Report 2020: Fat measurements via wet:dry weight ratios have continued throughout the 2019 season in fish lethally sampled, as well as non-lethally via the fat meter.

(12) A non-lethal intestinal wash will then be taken for microbial DNA extraction and sequencing. Water and control samples will also be taken to monitor environmental microbiome fluctuations as well as potential contamination
• Seasonal assessments will commence autumn 2017

• 2018 Report - Rather than non-lethal sampling we have been undertaking comprehensive dissections of individual fish

• Report 2019: Water samples have been gathered from all sites (stream, hatchery, aquarium, sea cages) at regular intervals.

• Report 2020: Environmental samples, including from water and insect biomass, have been taken on a weekly basis. Fish dissections continue

(13) Profiling of the gut microbiota will be via amplification and sequencing of the 16S v4 region on an Illumina MiSeq platform at the CGR Liverpool
• Scheduled autumn 2017.

• 2018 Report - First round of sequencing will be targeted at the University of Glasgow cohort of Burrishoole fish in March 2018. 16S profiling of gut communities from the rough river at Burrishoole will commence in April 2018 based on samples collected during spring / winter sampling 2017/18.

• Report 2019: This work is ongoing. Samples from WO, FO and RO fish held at the University of Glasgow aquaria have been sequenced. Samples from WO, FO and RO fish from Ireland have undergone library preparation and sequencing will commence in spring 2019.

• Report 2020: 70% of Sequencing libraries have been sent to Novogene for Illumina Novagene amplicon sequencing as well as PacBio full length amplicon seq for a subset to improve taxonomic identification. We await results in the next thee weeks. Aquarium sequencing in Glasgow is complete.

(14) Microbial amplicon sequence will be analysed using standard and custom workflows and correlated with fish growth and metabolic traits
• Scheduled autumn 2017.

• 2018 Report - At the UoG, workflows are now in place for amplicon sequencing based on SalmoSim farmed fish gut analyses. Metabolic covariates are those collected during respirometry and other energetic measurements (i.e. energy harvest from feeds, conversion efficiency).

• Report 2019: Growth and metabolic traits have been collected from fish in the Glasgow aquaria, Burrishoole hatchery, stream system and the sea cage system. Energy harvest from feeds, conversion efficiency have been measures from Glasgow aquaria fish in 2018.

• Report 2020: All Glasgow aquarium fish microbiomes have been fully analysed. Clear correlations between SMR and microbial composition have been uncovered across two biological replicate experiments, confirming a robust effect. A manuscript is in prep.


(15) To evaluate how these relationships change with season and how gut microbiota predict long-term performance, measurements of metabolism and microbial sampling will be repeated in late winter, while survival/growth will be assessed by five electrofishing surveys over 18 months (initial sampling Autumn, followed in the Winter, Spring, Summer and Autumn of the following year).
• Scheduled autumn 2017.

• 2018 Report - We are currently establishing the respirometry system for making measures of metabolic potential, which has taken slightly longer than expected to set up. In November 2017 we sampled five fish from the river for microbiome sampling. We also sampled 5 stocked ranch fish for microbiome analysis in November. In January 2018 we sampled a further 18 0+ salmon from the experimental population and 5 further ranch origin fish were sampled after respirometry.

• Report 2019: This work is ongoing. Sampling expeditions occurred in June and November 2018.

• Report 2020: This work is ongoing, samples have been sent for sequencing.

(16) All fish recaptured at the final sampling will be euthanized and their alimentary canal (cardiac
stomach, the pyloric caeca, mid-intestine, posterior intestine) removed for epithelial histology
('villus' -- salmon have densely folded columnar epithelia rather than villi -- density and folding) and morphometric measurements (relative total gut length, pyloric caecum fold number).
• Scheduled autumn 2017.

• 2018 Report - Samples are being routinely collected at the MI Burrishoole and guts collected in the appropriate buffers prior to shipment to Glasgow. At Glasgow, Pei Yang has been recruited to oversee histological analyses including Fluorescence In Situ Hybridization to identify the distribution of different microbes in respect of different gut structures.

• Report 2019: Similar activities in this context are underway in 2018 as occurred in 2017.

• Report 2020: Work is ongoing. A manuscript describing the FISH results is now in draft.

(17)Juvenile WO salmon brought as eggs from the experimental families produced at the
Burrishoole hatchery will be reared on a commercial pelleted food at the aquarium facility in
Glasgow.
• The transfer of eggs is scheduled for mid-February 2017.

• 2018 Report - Eggs from the experimental population established in Newport were successfully transferred to Glasgow in February 2017. These parr are now 20-40 g in weight. An addition subset of experimental eggs from the 2017/18 cohort will be transferred from the Newport facility to Glasgow in February 2018.

• Report 2019: Complete

(18) At 6 months of age fish (n=90, with 6 fish from each of 15 families) will be transferred to
90 individual flow-through aquaria, which will share a common water source and be held at
constant temperature (13°C). They will be fed a fixed ration (1% body mass); after a month-long period of acclimatization, their growth efficiency will be measured, together with SMR and AS
• Scheduled autumn 2017.

• 2018 Report - Two range finding experiments have already been carried out on two cohorts of stock parr to identify optimal conditions for the aquarium study. Fish are being left until 12 months to represent the optimal size for the respirometry chambers. The aquarium study will now commence on the first of March 2018 and will feature 20 individuals per group.

• Report 2019: Complete

• Report 2020: Manuscript in prep


(19) To test digestive efficiency, comparative bomb calorimetry of each controlled food
ration and faeces will be monitored over a two day period. Calorific measurements will be repeated in triplicate for each individual fish.
• Task scheduled to commence autumn 2017.

• 2018 Report - Faecal samples from range finding experiments have been submitted for bomb calorimetry at the University of Stirling. We are awaiting data.

• Report 2019: Complete

• Report 2020: Complete, ms in prep

(20) Faecal sampling, microbial diversity profiling and gut morphological analysis will also be carried out. Microbiota in water and feed will be 16S profiled as controls.
• Task scheduled to commence autumn 2017.

• 2018 Report - 16S amplification and library preparation from range finding experiments is complete and these samples are awaiting sequencing at the University of Glasgow.

• Report 2019: Complete

• Report 2020: Complete, ms in prep




(21) Eggs from each of the WO and FO populations will be transferred to the Nofima research facility (rented by Marine Harvest) in Norway, for on-growing to the marine stage of
development. 100 fish from each population (WO and FO) will be transferred to salt water test facilities where equivalent experiments to those described in Activity 1b will take place in marine phase post-smolts (300-500g).
• The transfer of eggs is scheduled for mid-February 2017. However we will now be transferring the eggs to the Marine Harvest's experimental facility at Ardnish, Lochailort, , Scotland rather than NOFIMA Norway.

• 2018 Report - The transfer of eggs to the NOFIMA research facility in Norway was scheduled for mid-February 2017. Subsequently it was planned to transfer the eggs to the Marine Harvest experimental facility at Ardnish, Lochailort, Scotland rather than NOFIMA Norway. Both plans have now been superseded by a new arrangement with the Marine Institute enabled by the establishment of their new marine research site at Bertreach Bui in Connemara. We have established two experimental populations in the hatchery. The first is an opportunistic study and consists a group of pure farm Mowi strain fish (n=5,083) and a group of captive bred Burrishoole ranch strain fish (n=4,751). These fish were introduced into sea cages as S1/2 in December 2017 having been subject to light manipulation to advance their physiological development. As a control prior to their introduction to the sea cage in 6 December 2017 microbiota were sampled from 22 individuals in addition to 12 individuals for fatty acid profiling. Subsequently we have been undertaking regular sampling of individuals (6 samples per visit) in the sea cages 18, 22 and 27 of December and most recently on the 5 January and 26 January. A full dissection has been undertaken on these fish including gut microbiota from stomach, pyloric caecae, mid gut and hind gut. In addition environmental water samples were also collected.For the scheduled study in the proposal approximately 1,000 swim up fry from each of the three experimental groups, farm origin, wild origin and captive bred ranch origin were transferred to the main hatchery facility at Burrishoole in March 2017. In September n=15 parr were sampled from each of the experimental groups for microbiota profiling in order to establish a baseline of the gut bacterial communities and stored at -80oC. These were on-grown to mid-October 2017 when 100 parr from each of the experiment groups were individually PIT tagged and measured, weight and length, to facilitate, monitoring of growth, transferred into three new tanks to provide triplicate equal mixtures of the experimental groups, each tank now consisting of 300 parr. In mid-January 2018, 27 fish, 9 of each experiment group identified on the basis of their PIT tag were sampled (i.e. 3 x9). The experimental hatchery population are scheduled to be introduced to the sea cages in mid-April 2018.

• Report 2019: In May 414 farmed, 603 ranched, 516 wild and a mixed group of PIT tagged smolts were introduced into four sentinel cages in the sea farm. The sea farm was sampled on 17 separate occasions. Fish were transported to the research facility in Newport for metabolic potential measurements and gut acquisition. 214 intestinal tracts were sampled from the sea farm, 42 from the s1/2 study and 172 from the S1 study. The metabolic rate of 55 fish were also phenotyped using ventilation rate as a proxy for SMR. The 2018 sea farm study was terminated on the 3 September 2018. A second hatchery based experimental population was ongrown throughout 2018. In contrast to the 2017 study this population has reciprocal hybrids. The hatchery population was sampled at the egg stage, alevin stage, and as parr up to the end of the year. It planned to transfer these fish to sea in April 2019.

• Report 2020: Further fish, including FO, WO and HO fish were put out to sea in May 2019. This sea cage trial is now complete. Full respirometry via was deployed to calculate SMR and AS on 200 fish. Microbiome samples were taken and have been sent for sequencing.




(22) Gut compartments will be set up across three linked Applikon bioreactors based at the University of Glasgow, School of Engineering (SoE). At the core of each Applikon bioreactor are six adjustable control loops to set basic parameters like temperature, pH, dissolved oxygen, agitation, level and foam.

• Establishment of the bioreactors is scheduled for mid-2017. However extensive testing is ongoing.

• 2018 Report - Gut compartments will be set up across three linked Applikon bioreactors based at the University of Glasgow, School of Engineering (SoE). At the core of each Applikon bioreactor are six adjustable control loops to set basic parameters like temperature, pH, dissolved oxygen, agitation, level and foam. All basic parameters have been established. The bioreactors are now connected and the first run is underway and scheduled for completion on the 21st of Feb. Live stream to monitor foam over on reactors can be found here: https://www.youtube.com/watch?v=hov_eaBUWOA



• Report 2019: Bioreactors have been working continuously throughout 2018 and the system is now fully validated. Communities of microbes in the reactors are now stable with respect to the gut communities of the host. Even most fastidious microbes found in the salmon gut (e.g. Mycoplasma) can be maintained now in the system.

• Report 2020: Bioreactors continued to run throughout 2019 on the validated system. A change in bioreactors conformation was also validated to improve biological replication. A manuscript is in prep to describe the validation experiment and samples sent for sequencing.


(23) Homogenised, filtered samples of different salmon gut compartments will be subjected to proteomics analysis via the University of Glasgow PolyOmics centre using tandem
liquid chromatography (LC), high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to identify major digestive enzyme classes present. A similar system will be applied to smaller non-volatile digestive secretions (e.g. bile salts). Once protein and bile salts are identified, in-house HPLC at the SoE can be used to differentially assess abundance. Volatile components of intestinal contents, especially short chain fatty acids (SCFAs), can be identified in house at the SoE using atandem gas chromatography (GC)-MS system. Commercial availability of the various enzymes will be assessed to identify the 'best match' in terms of function and activity for inclusion in the model.
• Scheduled sampling will commence mid-2017.

• 2018 Report - Proteomics and metabolomics are now complete. Biochemical marker identification is underway. Several key markers of microbial metabolism have been identified, including VFAs, lactate and others. We can now assess the abundance of these secondary metabolites directly via HPLC from faecal samples. We now use purified salmon bile to supplement the reactors.

• Report 2019: We now regularly use a GC-FID to undertake characterization of volatile microbial metabolites.

• Report 2020: We continue to use GC-FID on gut content samples.



(24) In addition to establishing the correct physico- and biochemical conditions, we propose to
introduce artificial mucosal surfaces for bacterial colonisation. Microbial classes in fish intestines are broadly separated into 'adherent' and 'non-adherent'. In common with most vertebrates, salmon mid- and hind-guts possess a thick lining of mucous secreted by goblet cells embedded in the columnar epithelium. After [21] we will therefore introduce beads (AnoxKaldnes K1 carrier, AnoxKaldnes, Sweden) coated with nutrient-free agar incorporating mucins purified from salmon intestine waste (Marine Harvest).
• Introduction of the beads is scheduled for mid-2017.

• 2018 Report - We have adopted the approach described in the project. The K1 beads are coated in mucin-infused agar and are currently floating in the pyloric caecum compartment medium. At the end of the current run, these K1 beads will be sliced, fixed and stained to visualise the distribution and diversity of colonising microbes by comparison to those known to be associated with the gut epithelium. To validate our model system, we will introduce selected fresh and saltwater salmon microbiota into our system and allow these to establish over a 48-hour cycle.

• Report 2019: Complete.


(25) To validate our model system, we will introduce selected fresh and saltwater salmon microbiota into our system and allow these to establish over a 48-hour cycle.
• The validation of freshwater salmon microbiota is scheduled for mid-2017. The validation of marine microbiota is set for mid-2018.

• 2018 Report - Our primary focus has been on the marine microbiome to date. Gut microbes have been seeded in from marine phase post-smolts. We intend to trial the freshwater gut model in Autumn 2018.

• Report 2019: Pushed out to 2019.

• Report 2019: Pushed out to mid 2020


(26) Based on field observations in field experiments, and a small number of metagenomic
sequencing runs from early gut simulator runs, we will develop 16S v4 primers targeted at phylum and family level components of the microbiome for qPCR This approach will allow the rapid monitoring of changes in abundance of core taxonomic classes over the course of the experiment. Validation of the system will occur when we can recover matching abundance profiles for core microbiota taxa between the original sample and bioreactor samples (especially the hind-gut compartment) at the end of the cycle.
• The development of the primers and their validation is scheduled for mid-2017

• 2018 Report - We have identified 8 taxon-specific primers so far that we will deploy to track our communities at a phylum and species level. In addition we have developed functional qPCR markers to target microbial processes identified from the metabolomic data - methanogensis, acetogenesis etc.

• Report 2019: Complete

(27) Microbiota cryopreserved from WO field and WO laboratory fish found to have extremes of SMR and AS, in both saltwater and freshwater life stages, will be selected for testing in the model system. Our target is to explant a total 10 microbiomes from salt and freshwater fish, respectively (20 total) into the model.
• This task is scheduled to follow on from flow respirometry measures in river, which will commence October 2017.

• 2018 Report - This activity will occur once artificial system is stabilized - likely to occur early 2019

• Report 2019: Planned for 2019.

• Report 2020: Pushed out to mid 2020


(28) Digestive characteristics of each microbiome (multiple biochemical markers of bacterial protein, carbohydrate and fat fermentation efficiency, monitored using HPLC and GC at 6-hour intervals, including ammonia, amines, phenols, sulphides, SCFAs, lactate and short chain oligosaccharides) will be measured in each of three independent cycles of each model system
• These measures are scheduled for mid-2017.

• 2018 Report - Key markers of microbial metabolism have been identified using metabolomic and metagenomic data acquired from analyses of marine phase salmon guts, as described above. Markers, include VFAs, lactate and others. We can now assess the abundance of these secondary metabolites directly via HPLC based on material that we are systematically sampling from the reactors.

• Report 2019: Abundances of these metabolites has been assessed using GC FID

• Report 2020: Abundances of these metabolites continue to be assessed using GC FID

(29) If links between the microbiota and metabolism of fish are found in living and ex-vivo systems, we propose to attempt some exploratory transplantation techniques in normal and broad-spectrum antibiotic treated juvenile salmon with the aim of establishing causation.
• The execution of this transplantation task will depend on the success of finding associations between the microbiota and the metabolism of the fish.

• 2018 Report - This task is scheduled for 2019.

• Report 2019: The work is scheduled for 2019 and will also involve direct fish-to-fish transplantation.

• Report 2020: This work is schedule for mid 2020



(30) We propose to undertake a pilot laboratory transfer experiment whereby energetically-divergent microbiota (originally from high and low energy phenotype fish) from the model system will be reciprocally transplanted via gavage into a cohort of 60 salmon parr (30 'low' energy fish (with low SMR, small AS, low nutritional energy harvest) and 30 'high' energy fish (with the opposite traits). A further 90 parr will act as controls: 30 to receive a sterile gavage, and 60 matching transplants (e.g. low energy microbiome to low energy fish) for comparison with reciprocal transplants. The 150-fish cohort will be followed to detect potential changes in energetic profile.

• This task will start in early 2018.

• 2018 Report - This task will now start in early 2019.

• Report 2019: As above, work is scheduled for 2019 and will also involve direct fish-to-fish transplantation.
• Report 2020: This work is schedule for mid 2020
Exploitation Route The draft artificial gut system will be of use to multiple stakeholders in the salmonid aquaculture industry

Understanding of the link between gut microbes and fish performance will also beef use to multiple stakeholders in the salmonid aquaculture industry
Sectors Agriculture, Food and Drink

 
Description We are now 75% into our project - and making excellent progress, with one associated publication published and several in prep. The artificial salmon gut system developed as part of this project - SalmoSim, is now validated and ready to address applied and fundamental research questions - feed activity, drug stability, probiotic efficacy etc. We continue to have regular interactions with industry partners Marine Harvest and Alltech in the form of meetings, visits, farm visits, and researcher exchanges. We have also had interest from other industrial partners in our system - including researcher and knowledge exchange with Cargyll and Calysta - both salmon feed manufacturers. Early data - currently in preparation for publication - suggests a clear link between microbial community diversity and metabolic rate in salmon. Down the line, we hope to be able to inform our industry collaborators and the wider nonacademic community of the role of different microbes in salmon digestion in metabolism and performance.
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Title A draft model of the Salmosim Gut Microbiome Simulator 
Description A central aim of the project was to develop a gut Microbiome by simulator. An early draft of the simulator is now available. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2018 
Provided To Others? No  
Impact it is currently too early to tell what impact this system will have. 
 
Title A large panel of tissue and gut content samples from farmed atlantic salmon 
Description We now have a large panel of microbial DNA samples collected from the intestines of farmed Atlantic salmon. 
Type Of Material Biological samples 
Year Produced 2017 
Provided To Others? No  
Impact this collection of samples represents a unique resource for understanding the microbiology of farmed Atlantic salmon. 
 
Title A panel of live bacterial strains isolated from the gut of farmed Atlantic salmon. 
Description Over 50 different microbial strains have been isolated and characterised the species level. these are currently stored at the University of Glasgow. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact We cannot yet foresee the impact of this collection of strains 
 
Description Collaboration with Industry Partner Alltech 
Organisation Alltech
Country Global 
Sector Private 
PI Contribution We established this partnership at the outset of the project. We are establishing an artificial salmon gut system to understanding the impact of different feed additives on salmon health.
Collaborator Contribution Our partners are providing expertise and training in microbiology, as well paying for a full time PhD student.
Impact None so far with the exception of knowledge exchange
Start Year 2017
 
Description Collaboration with University College Cork (UCC) and Marine Institute (MI) Ireland 
Organisation Marine Institute
Country Ireland 
Sector Academic/University 
PI Contribution The UCC and MI collaboration are a core component to this project - which represents a joint Science Foundation Ireland / BBSRC endeavor. Full detail of this partnership are included in the award document. The experimental stream system around which a large portion if the award is based is located in a field station in Burrishoole, Co. Mayo Ireland and is operated by MI and UCC. The University of Glasgow (UoG) provides expertise and training in salmonid physiology, microbiology, metagenomics and bioinformatics to >8 PhD students and 4 post doctoral resarchers based at Burrishoole.
Collaborator Contribution UCC/MI provide expertise is salmonid ecology, biology and genetics - providing training to 5 UoG PhD students and 2 post-docs. UCC/MI also contribute substantial research infrastructure in terms of the catchment, trap system, hatchery, marine cages and experimental stream.
Impact Several important publications: e.g. 10.1038/s41598-018-19323-z
Start Year 2013
 
Description Collaboration with industrial partner Marine harvest 
Organisation Marine Harvest
Country Norway 
Sector Private 
PI Contribution The University of Glasgow research team established a collaboration with Marine harvest. We are contributing knowledge and expertise around the influence of gut microbes on salmon gross and performance. In addition we are developing an artificial guts system that can act as a testbed for novel seeds and seed formulations.
Collaborator Contribution Marine harvest is providing expertise and training to University of Glasgow staff and students. Marine harvest is also providing marine cage facilities for rearing Salmon to undertake experiments as part of this project.
Impact Current outputs are principally focused at knowledge exchange
Start Year 2017
 
Description Interview for National News (BBC Radio Scotland) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact I was interviewed about the project on BBC Radio Scotland
Year(s) Of Engagement Activity 2017
URL https://www.bbc.co.uk/radioscotland
 
Description Series of short films about the project posted on youtube 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact With our Irish Project partner we have embarked on a series of videos presenting our work
Year(s) Of Engagement Activity 2018
URL http://www.youtube.com/watch?v=VCoahlcxN9w&feature=share