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Background: During spring and summer season shells of farmed mussels are targeted as a growth substrate by biofouling organisms such as barnacles (Balanus sp.) and tube worms (Pomatoceros triqueter). While barnacles can be removed from mussel shells relatively easily, tube worms are much more difficult and costly to remove. The affected mussels are often discarded due to concerns that damage to the vacuum packaging will occur and also because the affected mussels are much less desirable to consumers (Watson et al., 2009). The presence of the tube worms does not cause the mussels to be inedible, but their appearance and smell when cooked makes the mussels unappetizing to the consumer. While there is little evidence to suggest that the growth of these organisms on the mussel’s shells has any detrimental effect on the overall health of the mussels themselves or the quality of the meat, some studies suggest that biofouling may in some cases results in growth and weight reductions as well as reduced shell growth (Sievers et al., 2013). Scotland’s mussel farming industry operates at a high standard of quality, meaning that any mussels that has over 7% of its shell affected by fouling, is not considered to be of marketable quality (Kelly, 2002). Some estimates have suggested that the financial losses realised from tubeworm biofouling could be as high as ?500,000 per annum (Devlin, 2020). However, if the industry’s ambition to get to 18,000 tonnes production by 2037 is realised and the economic impacts of biofouling are not mitigated, this problem will become proportionally more significant, effectively acting as a drogue on future industry’s performances. The current control measures that are in place at mussel farms to try to counteract the problem of mussel fouling by these tubeworms mainly involves maintaining clean shells through the use of avoidance strategies (Watts et al., 2015). This includes attempting to time the shell cleaning process to coincide with the point of early attachment of the tubeworm larvae to the mussel shells. The shell cleaning process can involve subjecting the mussels to exposure to the air, washing, or cleaning using acetic acid, brine or hot water (Sievers et al., 2014). The tube worms can be removed from the shells during the early attachment phase to the shells without great difficulty. Although, this process is not cost effective and involves raising the mussels from the loch-depths to which they have become acclimatised, at a crucial point during their growth. This could have detrimental effects on the mussel’s rate of growth. Therefore, mussel farmers want to ensure that they undertake this task as few times as possible, ideally only once per growing season. In order for these preventative measures to be the most effective, the mussel farmers need to attempt to predict when the tube worm larvae will no longer be present in the water column, as this would be when the larvae have begun to attach to the mussel shell surface. The strategies used to do this currently involve monitoring water temperatures and weather patterns in order to estimate when the post-settlement fouling will occur (Sievers et al., 2014). However, these methods are not exact and involve some degree of guess work on the farmers part, ultimately resulting in unpredictable results and the above mentioned economic damage. This project is focused on developing and implementing a molecular assay which can identify the presence of Pomatoceros triqueter DNA either within environmental (plankton) samples or from surface swabs of mussel shells. This will result in the development of a P. triqueter monitoring capability which mussel farmers could utilise to optimise shell cleaning strategies in line with local fouling conditions to ultimately improve stock management and product quality.

Research Approach: The overarching aims of the proposed project are: A.The development of an optimised rapid diagnostic tests to identify the presence of P. triqueter DNA in plankton and shell swab samples. B.To identify and optimise the most applicable quantification methods C.To apply the newly developed test to identify the optimum timing for shell cleaning operations at commercial mussel farms. D.To verify the hypothesis that by implementing shell cleaning operations in accordance with the test results significantly reduces biofouling on mussel shells. Over the past year the Institute of Aquaculture has developed a preliminary DNA meta-barcoding tool for the identification of P. triqueter in plankton and mussel shell swab samples (Develin, 2020). Furthermore, when this tool was applied over the spring and summer period at two commercial mussel farming sites in the west coast of Scotland we were able to broadly estimate the timing of the shift from planktonic to benthic life stages of the target species, providing proof of concept of the proposed strategy (TRL 5). Molecular diagnostic tools are not uncommon in the aquaculture sector however they are usually directed towards pathogen detection in the finfish sector. Their use within shellfish production is less well established, not just in Scotland but globally. By their design such assays have high specificity and sensitivity which is essential for a field monitoring service. This project would develop the crude assay used in the proof of concept to establish a truly quantitative qPCR assay and validate the accompanying sample collection methodologies. As such this project will represent a clear example of effective technology transfer between the two most significant aquaculture sectors in Scotland and it will enable the Scottish shellfish industry to lead the way in the application of industry lead innovation on the global stage. During this project we will work in close collaboration with mussel industry operators in Shetland who will adhere to a rigorous sampling strategy to collect plankton and swab samples during the spring and summer months over two consecutive years at two commercial farm sites. Furthermore, temperature and salinity will be monitored by multi-parametric sensors and natural settlement of tube worms will be monitored on experimental settlement substrates deployed at the same sites. During the first year plankton and shell swab samples will be collected fortnightly and shipped to the IoA on a monthly basis alongside pictures of the experimental settlement substrates according to established methodology (Campbell and Kelly 2002). The samples will then be used to optimise the qPCR based assay and gather environmental and recruitment dynamic data at the experimental sites. The information acquired in the first year will inform the 2nd year data collection approach and crucially be used to dictate “intervention thresholds” based on the cross validation of observed biofouling and molecular quantitation. It is envisaged that in the second year sampling will take place at weekly intervals, focused around P. triqueter settlement period. This will allow for the identification of the time when the majority of tubeworms larvae would have settled and therefore indicating when shell cleaning operations should take place. At this point a portion of the mussel lines will be cleaned and re-deployed (treated lines) and part will not receive cleaning treatment (control). The treated and control lines will then be monitored for the following three months to verify whether a significant reduction in biofouling had taken place. The outcome of this project is the development and implementation of a molecular based P. triqueter detection assay to inform when on farm biofouling interventions should be performed. If implemented successfully this work will help realise a significant reduction in downgrading at harvest due to biofouling. In so doing this work will allow the Scottish shellfish industry to acquire reliable and data-based information to support critical decisions during the production cycle by providing a tool similar to those applied by the Scottish Salmon industry for the identification of pathogens (e.g. gill swab surveys for Amoebic Gill Disease). Furthermore, once developed, this tool could provide further critical support to the sector during the site selection process allowing farmers to determine the abundance of P. triqueter biofouling at prospective sites during the early site survey stages.