In-situ molecular-based monitoring: a tool for tackling the operational and environmental challenges of aquaculture

This innovative project will deliver a technology enabling better control of the disease- and environmental challenges posed by aquaculture intensification. The Norwegian aquaculture industry has a significant socioeconomic value. However, the planned intensification of fish production from aquaculture will require improvement of production sustainability as a response to increase environmental pressure. Today, parasitism and diseases, causing significant fish production loss for this industry, are mitigated using tons of chemicals every year that causes environmental concerns. Early detection of fish infection is one solution to these challenges, providing farmers with timely critical information. Using environmental DNA (eDNA) is seen as one way to inform about critical occurrence of pathogens to the farm operators. ISMOTOOL will use a ?genosensor?, the Environmental Sampling Processor (ESP), to automatize sampling and perform in-situ analysis of water samples for early detection and quantification of fish pathogens. The ESP developed at MBARI (USA) is a compact and fully robotized underwater, true ?lab in a can? that processes seawater samples for specific DNA-based detection of marine organisms and can communicate the results remotely to the end-users for decisions. Target organisms of concern for Atlantic salmon are the salmon louse (Lepeophtheirus salmonisi) and microorganisms like Paramoeba perurans, the pathogen causing amoebic gill disease (AGD). Fish escapes also requires better control and early detection. For this, ESP will target eDNA shed naturally from salmon (and trout) to potentially use for fish escapees. Within WP1, the number of marker gene copies (both CO1 and 16SrRNA mitochondrial) was quantified for different number of individuals (1-10) for free-swimming stages (naupli and copepodites) of L. salmonis. Quantitative PCR was performed at three sampling periods (October 2019, May and September 2020) at a fish farm in Western Norway and samples collected at four locations around and three different depths (1m, 5m and 10m). Overall, detection was higher in May and September 2020, lower in October 2019. We selected the 16S rRNA mitochondrial DNA of L. salmonis to analyze the samples but we also used an assay previously reported in literature that targets both L. salmonids and Caligalus elongatus sealice species. The performance of the assay developed in this project was better. Our assay is also more specific for L. salmonids detection. In WP2, we tested P. perurans eDNA detection and quantification in fish farms and infection trials. Here, eDNA samples were taken at the farm locations (see WP1) with a simulated EPS-method (0.5L water) and manual glassfiber filtration (5L water; GF-method). The samples were analyzed with simulated ESP-qPCR and standard qPCR, both P. perurans specific (previously developed in this project). P. perurans eDNA concentrations were highest at 5m depth in October 2019, and equally high at 5m and 10m depths in September 2020. In June 2020, P. perurans remained undetected. However, the ESP-method yielded lower eDNA concentrations than the GF-method, possibly resulting from filtration volume used. A new, virulent cell-line of P. perurans was established, and in ongoing infection trials we have shown that P. perurans eDNA in the water increases with increasing time after infection. For WP3, the ESP collected and preserved eDNA samples for post-deployment analysis and real-time analysis of species eDNA, including rainbow trout, which was detected twice during a 14-day deployment. New reagents that reduce issues with inhibition, increases sensitivity, decreases analysis time and improve analysis conditions have been tested and validated. A new system to hold our assay reagents onboard the ESP, which should help mitigate troubleshooting observed previously, was developed. We have now developed a sampling program for the ESP which is matched with the tide to allow collection of water both when the current transport eDNA away from and into the fish pens. This will help us assess both the current infection status of the fish pens when the water is outgoing and track potential escapees or potential incoming diseases when the water is ingoing. For all target organisms and assays, we have performed validation of ESP methods and manual methods, both for eDNA filtering and qPCR. In fall 2021 (16.10-13.11), we did a field test with ESP at 6 m depth, downstream a fish farm in western Norway. ESP used the assays (2 for sealice, 2 for AGD, 1 for Salmon fish) developed/tested earlier. Overall, both detection and quantified amount of eDNA fluctuated over the period: AGD and fish eDNA were detected in some samples, salmon lice was not. This was likely the results of several variables: current direction and strength, analytical volume (<150 ml per sample), distance to the nearest cage, season, and the downsizing of pens with salmon fish underway. Data will be analyzed further.

Prosjektets viktigste leveranse har vært å demonstrere at ESP-roboten faktisk fungerte for påvisning av fisk, AGD og lakselus i sanntid, samt å utvikle og teste miljø-DNA metodikk til overvåkning av AGD og lakselus, som er fiskeparasitter som forårsaker store tap i oppdrettsnæringen og medfører både velferdsproblemer og dødelighet for oppdrettsfisk. Ved å bruke miljø-DNA-analyser av vann slipper kan dette være en alternativ metode til å fange eller drepe fisken for overvåkningsformål. Metodene er kostnadseffektive og vil kunne et få bredt anvendelsesområde innen fiskeoppdrett, for å få tidsriktig informasjon om mulige smitte fra ulike agenser og dermed igangsette tiltak for å redusere påslaget, uten å belaste miljøet for mye.

The aquaculture industry is facing several environmental challenges, including disease, salmon lice invasions and escape of farmed fish. Meeting these challenges will require novel approaches and development of innovative tools. The Environmental Sampling Processor (ESP) is a molecular-based autonomous monitoring device designed to collect water samples, extract DNA from organisms in the water and perform in situ molecular analyses automatically. Two-way wireless communication makes it possible to transmit the results from the analyses back to the laboratory only within hours of sample, allowing for rapid (same day) actions to be taken. The aim of the proposed study is to use ESP for operational monitoring and fish welfare towards aquaculture fish production to improve sustainability and operations. This tool will be targeted to (1) fish pathogens and parasites in the water column, (2) escaped farmed fish in the marine environment, all posing present challenges for this industry. Four work packages (WP) are planned: In WP1-3, we will adapt analytical protocols of ESP to detection and quantification of (1) parasitic salmon louse (Lepeophtheirus salmonis) (WP1), (2) an agent of amoebic gill disease (AGD); Paramoeba peruransi (WP2), and (3) escaped farmed Atlantic salmon and rainbow trout (WP3). After optimizing the species-specific molecular assays using bench laboratory equipment compatible with ESP, we will evaluate these assays on the ESP itself, and perform a field experiment (WP4). Results obtained from ESP detection and quantification will be compared with those obtained by use of the current detection and monitoring methods. The ESP instrument is available for ISMOTOOL through collaboration with DHI, the only company in Europe providing an ESP instrument.