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Accurate, cost‐effective monitoring of fish is required to assess the quality of lakes under the European Water Framework Directive. Recent studies have shown that environmental DNA (eDNA) metabarcoding is an effective and non‐invasive method, which can provide semi‐quantitative information about fish communities in large lakes. This study further investigated the potential of fish‐based eDNA metabarcoding as a tool for lake assessment by collecting and analysing water samples from eight Welsh lakes and six meres in Cheshire, England, with well‐described fish faunas. Water samples (N = 252) were assayed using two mitochondrial DNA regions (Cytb and 12S rRNA). eDNA sampling indicated the presence of very similar species in the lakes compared to those expected on the basis of existing and historical information. Firstly, 24 species were detected, with a total of 111 species occurrences in the lakes studied using eDNA. Secondly, there was a significant positive correlation between expected faunas and eDNA data in terms of confidence of species occurrence (Spearman's r = 0.74, df = 109, p < 0.001). Thirdly, eDNA data can estimate relative abundance with the standard five‐level classification scale (‘DAFOR’). Lastly, four ecological fish communities were characterized using eDNA data which agree with the predefined lake types according to environmental characteristics. Synthesis and applications. There are some limitations when using conventional captured‐based methods for surveying species richness and relative abundance, such as morphological identification bias, difficulties in recording small‐bodied, rare and/or elusive species and destructive impacts on the environment. This study provides further evidence that environmental DNA metabarcoding outperforms other captured‐based survey techniques in a wide range of lake types for community‐level analysis whether in species detection, relative abundance estimate using the standard five‐level classification scale or characterization ecological fish communities. Therefore, the fish‐based environmental DNA metabarcoding, a non‐invasive genetic method, has great potential as an assessment tool for lake quality under the European Water Framework Directive. There are some limitations when using conventional captured‐based methods for surveying species richness and relative abundance, such as morphological identification bias, difficulties in recording small‐bodied, rare and/or elusive species and destructive impacts on the environment. This study provides further evidence that environmental DNA metabarcoding outperforms other captured‐based survey techniques in a wide range of lake types for community‐level analysis whether in species detection, relative abundance estimate using the standard five‐level classification scale or characterization ecological fish communities. Therefore, the fish‐based environmental DNA metabarcoding, a non‐invasive genetic method, has great potential as an assessment tool for lake quality under the European Water Framework Directive.

Determining the timing and location of fish reproductive events is crucial for the implementation of correct management and conservation schemes. Conventional methods used to monitor these events are often unable to assess the spawning activity directly or can be invasive and therefore problematic. This is especially the case when threatened fish populations are the study subject, such as the Arctic charr (Salvelinus alpinus L.) populations in Windermere (Cumbria, UK). Arctic charr populations have been studied in this lake since the 1940s, and the locations and characteristics of spawning grounds have been described in detail using techniques such as hydroacoustics, as well as physical and visual surveys of the lake bottom. Here, in conjunction with established netting surveys, we added an environmental DNA (eDNA) metabarcoding approach to assess the spatial distribution of Arctic charr in the lake throughout the year to test whether this tool could allow us to identify spawning locations and activity. Sampling was carried out between October 2017 and July 2018 at three locations in the lake, covering putative and known spawning sites. eDNA metabarcoding provided accurate spatial and temporal characterization of Arctic charr spawning events. Peaks of Arctic charr relative read counts from eDNA metabarcoding were observed during the spawning season and at specific locations of both putative and known spawning sites. Net catches of mature Arctic charr individuals confirmed the association between the Arctic charr spawning activity and the peaks of eDNA metabarcoding relative read counts. This study demonstrates the ability of eDNA metabarcoding to effectively and efficiently characterize the spatial and temporal nature of fish spawning in lentic systems. This study demonstrates the ability of environmental DNA (eDNA) metabarcoding to provide an accurate spatial and temporal characterization of Arctic charr spawning events in a lake setting. We observed peaks of Arctic charr relative read counts from eDNA metabarcoding during the spawning season and at specific locations of putative and known spawning sites. Net catches of mature Arctic charr individuals confirmed the association between the Arctic charr spawning activity and the peaks of eDNA.

Environmental DNA (eDNA) metabarcoding is transforming biodiversity monitoring in aquatic environments. Such an approach has been developed and deployed for monitoring lake fish communities in Great Britain, where the method has repeatedly shown a comparable or better performance than conventional approaches. Previous analyses indicated that 20 water samples per lake are sufficient to reliably estimate fish species richness, but it is unclear how reduced eDNA sampling effort affects richness, or other biodiversity estimates and metrics. As the number of samples strongly influences the cost of monitoring programmes, it is essential that sampling effort is optimised for a specific monitoring objective. The aim of this project was to explore the effect of reduced eDNA sampling effort on biodiversity metrics (namely species richness and community composition) using algorithmic and statistical resampling techniques of a data set from 101 lakes, covering a wide spectrum of lake types and ecological quality. The results showed that reliable estimation of lake fish species richness could, in fact, usually be achieved with a much lower number of samples. For example, in almost 90% of lakes, 95% of complete fish richness could be detected with only 10 water samples, regardless of lake area. Similarly, other measures of alpha and beta-diversity were not greatly affected by a reduction in sample size from 20 to 10 samples. We also found that there is no significant difference in detected species richness between shoreline and offshore sampling transects, allowing for simplified field logistics. This could potentially allow the effective sampling of a larger number of lakes within a given monitoring budget. However, rare species were more often missed with fewer samples, with potential implications for monitoring of invasive or endangered species. These results should inform the design of eDNA sampling strategies, so that these can be optimised to achieve specific monitoring goals.

Invasive non-native species (INNS) are one of the main drivers of biodiversity loss globally, however to what extent INNS predators modify and utilise the invaded communities is still debated. This is particularly the case for arthropod INNS whose trophic interaction and predatory impact can be challenging to detect and describe. The application of DNA-based analysis, such as DNA metabarcoding, to the study of trophic interactions is often referred to as “MATI” (Molecular Analysis of Trophic Interactions). This approach has great potential in terms of increased sensitivity, higher resolution of prey identification and application to large-scale field studies, compared to previously established methods. This thesis focuses on describing trophic interactions in three non- native arthropod predators using this approach, with a key focus on intraguild predation (IGP), which has been shown to speed up invasion and facilitate establishment of invaders. In the first data chapter I focussed on the invasive amphipod, Dikerogammarus villosus, commonly known as “killer shrimp” to prove the concept that detection of prey DNA is possible in a controlled feeding experiment, and at a small field scale. From this proof of concept, I increased the scale of the field study by separately investigating the trophic interactions of three INNS: 1) D. villosus (Amphipoda: Gammaridae), 2) Harmonia axyridis (Coleoptera: Coccinellidae), and 3) the newly detected non-native Crangonyx floridanus (Amphipoda: Crangonyctidae). Data was collected for all species from UK sites across two seasons. The overarching goals were to investigate the broad trophic interactions of the target INNS across space and time, with a focus on detecting and understanding the importance of IGP in each target INNS. I predicted that high levels of IGP could be detected in all three target INNS. Firstly, I demonstrated that the method could detect prey species in both controlled and in field conditions, but detection success varied between prey taxa. I detected only low levels of IGP in D. villosus compared to a native amphipod, G. zaddachi, and found no evidence of IGP in H. axyridis, despite these species’ reputations as important IG predators. I relate this to the availability of IG prey in the wider community. By contrast I detected high levels of reciprocal IGP between the newly detected C. floridanus and the established, non-native C. pseudogracilis. Interestingly, IGP was asymmetric, in favour of the new invader, which could facilitate its establishment by eliminating competition. Together, these results demonstrate the applicability and also the challenges of DNA metabarcoding to molecular trophic interactions of INNS to understand the extent of their interactions in the invaded communities. I provide novel insight into the predatory dynamics of the three target species and their impact on the invaded communities.

Early detection is paramount for attempts to remove invasive non-native species (INNS). Traditional methods rely on physical sampling and morphological identification, which can be problematic when species are in low densities and/or are cryptic. The use of environmental DNA (eDNA) as a monitoring tool in freshwater systems is becoming increasingly acceptable and widely used for the detection of single species. Here we demonstrate the development and application of standard PCR primers for the detection of two freshwater invasive species which are high priority for monitoring in the UK and elsewhere: the Dreissenid mussels; Dreissena rostriformis bugensis (Andrusov, 1987) and D. polymorpha (Pallas, 1771). We carried out a rigorous validation process for testing the new primers, including DNA detection and degradation experiments in mesocosms, and a field comparison with traditional monitoring protocols. eDNA from single individuals of both mussel species could be detected within four hours of the start of the mesocosm experiment. In field trials, the two mussel species were detected at all sites where the species are known to be present, and eDNA consistently outperformed traditional kick-net sampling for species detection. These results demonstrate the applicability of standard PCR for eDNA detection of freshwater invasive species.

Early detection is paramount for attempts to remove invasive non-native species (INNS). Traditional methods rely on physical sampling and morphological identification, which can be problematic when species are in low densities and/or are cryptic. The use of environmental DNA (eDNA) as a monitoring tool in freshwater systems is becoming increasingly acceptable and widely used for the detection of single species. Here we demonstrate the development and application of standard PCR primers for the detection of four freshwater invasive species which are high priority for monitoring in the UK and elsewhere: Dreissenid mussels; Dreissena rostriformis bugensis (Andrusov, 1987) and D. polymorpha (Pallas, 1771), and Gammarid shrimps; Dikerogammarus villosus (Sowinsky, 1984) and D. haemobaphes (Eichwald, 1843). We carried out a rigorous validation process for testing the new primers, including DNA detection and degradation rate experiments in mesocosm, and a field comparison with traditional monitoring protocols. We successfully detected all four target species in mesocosms, but success was higher for mussels than shrimps. eDNA from single individuals of both mussel species could be detected within four hours of the start of the experiment. By contrast, shrimp were only consistently detected at higher densities (20 individuals). In field trials, the two mussel species and D. haemobaphes were detected at all sites where the species are known to be present, and eDNA consistently outperformed traditional kick sampling for species detection. However, D. villosus eDNA was only detected in one of five sites where the species was confirmed by kick sampling. These results demonstrate the applicability of standard PCR for eDNA detection of freshwater invasive species, but also highlight the importance of differences between taxa in terms of the detection ability.

Determining the timing and location of fish reproductive events is crucial for the implementation of correct management and conservation schemes. Conventional methods used to monitor these events are often unable to assess the spawning activity directly or can be invasive and therefore problematic. This is especially the case when threatened fish populations are the study subject, such as the Arctic charr (Salvelinus alpinus L.) populations in Windermere (Cumbria, UK). Arctic charr populations have been studied in this lake since the 1940s, and the locations and characteristics of spawning grounds have been described in detail using techniques such as hydroacoustics, as well as physical and visual surveys of the lake bottom. Here, in conjunction with established netting surveys, we added an environmental DNA (eDNA) metabarcoding approach to assess the spatial distribution of Arctic charr in the lake throughout the year to test whether this tool could allow us to identify spawning locations and activity. Sampling was carried out between October 2017 and July 2018 at three locations in the lake, covering putative and known spawning sites. eDNA metabarcoding provided accurate spatial and temporal characterisation of Arctic charr spawning events. Peaks of Arctic charr read counts from eDNA metabarcoding were observed during the spawning season and at specific locations of both putative and known spawning sites. Net catches of mature Arctic charr individuals confirmed the association between the Arctic charr spawning activity and the peaks of eDNA metabarcoding read counts. This study demonstrates the ability of eDNA metabarcoding to effectively and efficiently characterize the spatial and temporal nature of fish spawning in lentic systems. Competing Interest Statement The authors have declared no competing interest.

Abstract Fishes stocked for recreation and angling can damage freshwater habitats and negatively impact biodiversity. The pond-associated crucian carp (Carassius carassius) is rare across Europe and stocked for conservation management in England, but impacts on pond biota are understudied. Freshwater invertebrates contribute substantially to aquatic biodiversity, encompassing many rare and endemic species, but their small size and high abundance complicates their assessment. Practitioners have employed sweep-netting and kick-sampling with microscopy (morphotaxonomy), but specimen size/quality and experience can bias identification. DNA and eDNA metabarcoding offer alternative means of invertebrate assessment. We compared invertebrate diversity in ponds (N = 18) with and without crucian carp using morphotaxonomic identification, DNA metabarcoding, and eDNA metabarcoding. Five 2-L water samples and 3-minute sweep-net samples were collected at each pond. Inventories produced by morphotaxonomic identification of netted samples, DNA metabarcoding of bulk tissue samples, and eDNA metabarcoding of water samples were compared. Alpha diversity was greatest with DNA or eDNA metabarcoding, depending on whether standard or unbiased methods were considered. DNA metabarcoding reflected morphotaxonomic identification, whereas eDNA metabarcoding produced markedly different communities. These complementary tools should be combined for comprehensive invertebrate assessment. Crucian carp presence minimally reduced alpha diversity in ponds, but positively influenced beta diversity through taxon turnover (i.e. ponds with crucian carp contained different invertebrates to fishless ponds). Crucian carp presence contributes to landscape-scale invertebrate diversity, supporting continued conservation management in England. Our results show that molecular tools can enhance freshwater invertebrate assessment and facilitate development of more accurate and ecologically effective pond management strategies. Competing Interest Statement The authors have declared no competing interest. Footnotes * This version of the manuscript has been revised for resubmission to a journal. * https://doi.org/10.5281/zenodo.3993125

1. Accurate, cost-effective monitoring of fish is required to assess the quality of lakes under the European Water Framework Directive (WFD). Recent studies have shown that environmental DNA (eDNA) metabarcoding is an effective and non-invasive method, which can provide semi-quantitative information on fish communities in large lakes. 2. This study further investigated the potential of eDNA metabarcoding as a tool for WFD status assessment by collecting and analysing water samples from eight Welsh lakes and six meres in Cheshire, England, with well described fish faunas. Water samples (N = 252) were assayed using two mitochondrial DNA regions (Cytb and 12S rRNA). 3. eDNA sampling indicated the presence of very similar species in the lakes compared to those expected on the basis of existing and historical information. In total, 24 species were detected with a total of 111 species occurrences in the lakes studied using eDNA. Secondly, there was a significant positive correlation between expected faunas and eDNA data in terms of confidence of species occurrence (Spearman's r = 0.74, df = 109, p < 0.001). Thirdly, eDNA data can estimate relative abundance with the standard five-level classification scale (DAFOR). Lastly, four ecological fish communities were characterised using eDNA data which agrees with the pre-defined lake types according to environmental characteristics. 4. Synthesis and applications. This study provides further evidence that eDNA metabarcoding could be a powerful and non-invasive monitoring tool for WFD purpose in a wide range of lake types, considerably outperforming other methods for community level analysis. Footnotes * Figure 4 and Table 5 revised. Supplemental files updated.

Arginine uptake and yeast arginase activity were studied throughout the alcoholic fermentation of a white grape must ( Vitis vinifera L. cv Fiano), carried out by sequential inoculation ( Torulaspora delbrueckii and S. cerevisiae , TD-SC) and compared with a S. cerevisiae single fermentation (SC). In both samples, yeast assimilable nitrogen (YAN) consumption was mainly during the early phase of alcoholic fermentation (before S. cerevisiae addition in the sequential fermentation). T. delbrueckii alone and S. cerevisiae similarly metabolized YAN, which was further consumed in TD-SC sample following the S. cerevisiae inoculation. The only relevant arginine uptake was found about 3 days after the first inoculum (simultaneously with the greatest YAN consumption) and it appeared to be higher in SC (0.24 g/L) than in TD-SC (0.12 g/L). The kinetic parameters, estimated by means of the Hill equation, and in particular V max values (56.0 ± 1.6 U/mg BSAeq at 48 h for SC and 73.3 ± 2.7 U/mg BSAeq at 66 h for TD-SC) indicated the maximum arginase activity at the same point of time corresponding to the lowest YAN amount (48 h for S. cerevisiae and 66 h for T. delbrueckii ).