ABSTRACTSalmonid aquaculture, a major component of the Northern European, North American, and Chilean coastal economies, is under threat from challenges to gill health, many of which originate from plankton communities. A first step toward mitigating losses is to characterize the biological drivers of poor gill health. Numerous planktonic taxa have been implicated, including toxic and siliceous microalgae, hydrozoans, and scyphozoans; however, rigorous longitudinal surveys of plankton diversity and gill health have been lacking. In the current study, we present and assess an exhaustive identification approach combining both morphological and molecular methods together with robust statistical models to identify the planktonic drivers of proliferative gill disease (PGD) and fish mortality. We undertook longitudinal evaluation at two marine aquaculture facilities on the west coast of Scotland using daily data collected during the 2021 growing season (March–October). Examining these two different sites, one sheltered and one exposed to the open sea, we identified potentially new, important, and unexpected planktonic drivers of PGD and mortality (e.g., doliolids and appendicularians) and confirmed the significance of some established threats (e.g., hydrozoans and diatoms). We also explored delayed or “lagged” effects of plankton abundances on gill health and undertook a comparison of environmental DNA (eDNA) metabarcoding and microscopy in their ability to identify and quantify planktonic species. Our data highlight the diversity of planktonic threats to salmonid aquaculture as well as the importance of using both molecular and morphological approaches to detect these. There is now an urgent need to expand systematic longitudinal molecular and morphological approaches across multiple sites and over multiple years. The resultant catalogue of main biological drivers will enable early warning systems, new treatments, and, ultimately, a sustainable platform for future salmonid aquaculture in the marine environment.

ABSTRACTEnvironmental DNA (eDNA) consists of genetic material shed by living organisms, including those that are deceased, offering a unique opportunity to detect and identify terrestrial insect pests without requiring visual identification. The sweetpotato whitefly, Bemisia argentifolii, and the twospotted spider mite, Tetranychus urticae, are notorious for causing crop losses through virus transmission and direct feeding. Our study aimed to: (1) assess the effectiveness of B. argentifolii literature‐based PCR primers compared to newly developed primers for eDNA amplification, (2) evaluate the sensitivity of conventional PCR (cPCR) and real‐time quantitative PCR (qPCR) for detecting eDNA of B. argentifolii and T. urticae, (3) establish a rapid eDNA processing methodology using the LGC Biosearch Technologies QuickExtract DNA extraction kit and the Qiagen DNeasy Blood and Tissue kit, and (4) test the specificity of the developed primers against non‐target species. B. argentifolii and T. urticae were confined to tomato leaves (Solanum lycopersicum) using clip cages for 24 h, after which eDNA was collected from leaf surfaces using a water spray method, filtered, and processed for DNA amplification. While literature‐based primers showed sufficient sensitivity, their specificity for eDNA applications was inadequate, prompting the design of novel PCR primers for both pest species. Positive eDNA detection was achieved with both amplification methods, with qPCR proving more reliable than cPCR due to the latter's inconsistent performance with positive control samples. We also introduced a rapid eDNA processing approach using the QuickExtract DNA extraction kit, contrasting it with the more conventional Qiagen DNeasy Blood and Tissue kit. We believe that our findings are the first step toward the practical use of eDNA as a highly sensitive, early detection technique.

Abstract Marine mammal foraging grounds are popular focal points for marine protected area (MPA) implementation, despite being temporally dynamic, requiring continuous monitoring to infer prey availability and abundance. Marine mammal distributions are assumed to be driven by their prey in foraging areas, but limited understanding of prey distributions often prevents us from exploring how shifting prey availability impacts both seasonal and long‐term marine mammal distributions. Environmental DNA (eDNA) metabarcoding could enhance the understanding of marine mammal habitat use in relation to their prey through simultaneous monitoring of both. However, eDNA applications focused on marine mammals or predator–prey dynamics have been limited to date. In this study, we assess spatiotemporal changes in the distribution and abundance of cetaceans, minke whales ( Balaenoptera acutorostrata ), bottlenose dolphins ( Tursiops truncatus ) and harbor porpoises ( Phocoena phocoena ) in relation to key prey species in a newly established MPA, employing eDNA metabarcoding. We recovered 105 molecular operational taxonomic units (OTUs) from marine vertebrates using two primer sets targeting 12S and 16S genes, along with 112 OTUs from a broader eukaryotic primer set targeting 18S rRNA. Overall, key forage fish prey species, sandeels and clupeids, were the most abundant teleost fishes detected, although their availability varied temporally and with distance from shore. We also found clear spatial partitioning between coastal bottlenose dolphins and the more pelagic minke whales and harbor porpoises, paralleling availability of their main prey species. Other species of conservation interest were also detected including the critically endangered European eel ( Anguilla anguilla ), Atlantic bluefin tuna ( Thunnus thynnus ), and the invasive pink salmon ( Oncorhynchus gorbuscha ). This study demonstrates the application of eDNA to detect spatiotemporal trends in the occurrence and abundance of cetacean predators and their prey, furthering our understanding of fine‐scale habitat use within MPAs. Future long‐term monitoring of predator–prey dynamics with eDNA could improve our ability to predict climate‐induced shifts in foraging grounds and enhance rapid responses with appropriate management actions.

Abstract Environmental DNA (eDNA) degradation influences the effectiveness of eDNA‐based biodiversity monitoring, but the factors that determine the rate of decay of eDNA in terrestrial environments are poorly understood. We assessed the persistence of vertebrate eDNA from a mock vertebrate community created with soil from zoo enclosures holding 10 target species from different taxonomic classes (reptiles, birds, and mammals) and of different biomass (little penguin and giraffe). We examined species detection rates resulting from eDNA metabarcoding, as well as relative eDNA concentrations via qPCR, from soil samples over eight time points (0–12 weeks), during exposure to three ambient temperatures (10, 25, and 40°C) and three levels of ultraviolet B (UV‐B) radiation (0%, 50%, and 100% intensity). We recorded considerable variation in detectability between species, independent of temperature, and UV‐B effects. Quantitative polymerase chain reaction (PCR) indicated degradation of eDNA over time for all temperature and UV treatments, although it was still possible to detect eDNA from some species after 12 weeks. Degradation rates were lowest for high UV‐B treatments, presumably due to UV‐B reducing bacterial metabolism. The temperatures investigated did not influence eDNA decay. Our results indicate that eDNA in soil can persist under a range of temperatures and high UV radiation for longer than expected. Sheltered sites with minimal UV‐B radiation, which have previously been considered ideal sites for terrestrial eDNA collection, may not be optimal for eDNA persistence in some cases due to microbial decay. A better understanding of eDNA degradation in terrestrial environments is needed to enhance the accuracy of eDNA metabarcoding for surveying terrestrial vertebrate communities.

Abstract To reach the renewable energy targets set by the European Commission, a tenfold expansion of the installed offshore wind farms is needed. Since the construction of offshore wind farms may affect local soft‐sediment fauna, an efficient monitoring technique is needed to monitor the potential effects on the marine ecosystem. Here, we assess whether eDNA metabarcoding is a suitable alternative to monitor fish and epibenthos biodiversity in these difficult to access marine habitats. Water sampling and trawl surveys were conducted in parallel in 12 coastal and 18 offshore sites, the latter located inside and outside two offshore wind farms in the Belgian part of the North Sea. 12S eDNA metabarcoding retrieved 85.7% of the fish species caught in the beam trawls, whereas the COI eDNA metabarcoding only identified 31.4% of the epibenthic invertebrate species. Furthermore, the 12S marker resulted in an additional detection of 26 unique fish species, whereas the COI marker detected an additional 90 invertebrate species. Spatial patterns in alpha diversity recovered with eDNA metabarcoding were not significantly different from those observed with morphological determination. Significant differences were found in fish and invertebrate community structures between the coastal, transition and offshore zones as well as on the smaller wind farm scales, which agreed with the morphological beam trawl data. Indicator species found with morphological beam trawl monitoring for each of the three zones (coastal, transition, offshore) were also detected with 12S eDNA metabarcoding, and the latter method detected an additional 31 indicator species. Our findings show the need for adequate quality control of the obtained species lists and reveal that 12S eDNA metabarcoding analyses offers a useful survey tool for the monitoring of fish communities in offshore wind farms, but the used COI assay did not adequately capture the epibenthic communities as observed with beam trawl data.

Abstract Early detection of invasive species is crucial for effectively controlling the potential damage they can inflict on the ecosystems. In contrast to the many limitations that conventional detection methods like visual surveys and netting hold, environmental DNA assay provides a powerful alternative. This non‐invasive, highly sensitive, and user‐friendly technique offers the advantage of detecting invasive species even in areas where direct observation is challenging, thus addressing the shortcomings of traditional techniques and enhancing overall accuracy in estimating distribution. The spread of invasive Pterygoplichthys sp. has become a cause for concern in biodiversity‐rich countries like India. Despite this, comprehensive studies on the prevalence of this invasive species are limited. The Eastern Ghats of India remain under‐explored with a high potential for supporting diverse lifeforms. Studying the extent of biological invasions in the Eastern Ghats is essential for effective management and to mitigate the ecological and socioeconomic impacts of invasive species. In this study, we have designed and optimized an eDNA‐based quantitative PCR assay to map the presence and spread of invasive Pterygoplichthys sp. in selected freshwater ecosystems of the Eastern Ghats. This assay detected invasive Pterygoplichthys sp. in almost 65% of the total locations sampled. This study can be further extended to larger geographical areas, which can contribute to formulating necessary measures by the authorities to manage invasion and conserve the diversity of the freshwater ecosystem.

Abstract Fish aquaculture potentially affects the local ecosystem through feed (leftovers) and feces. However, evaluation of its impacts on biodiversity, especially those of mobile organisms such as fish, is not straightforward. Here, we report a case study in which environmental DNA (eDNA) metabarcoding with MiFish universal primers for the 12S rRNA region of fish is used to detect and assess the impact of aquaculture on local fish communities. In Shizugawa Bay, Minamisanriku Town, Miyagi Prefecture, Japan, Oncorhynchus kisutch (coho salmon) is aquacultured in sea cages from November to July, but is absent from August to October. We conducted eDNA surveys in June, August, October, and December 2021 of water at depths of 5 m and 1 m above the bottom obtained from two sites, one approximately 100 m and the other approximately 2500 m from the nearest aquaculture site. The total number of operational taxonomic units (OTUs) detected was 122, with a maximum of 50 and a minimum of 10. With distance‐based redundancy analysis (dbRDA) and PERMANOVA applied to the eDNA‐based community compositional data, we detected a significant effect of the presence of O. kisutch aquaculture on fish biodiversity. The generalized linear model (GLM) applied to each detected OTUs indicated that the aquaculture was associated positively with Leucopsarion petersii (ice goby), and Oncorhynchus spp. and negatively with Chaenogobius annularis (forktongue goby). Environmental DNA metabarcoding surveys, combined with multivariate statics, are a promising tool to identify the dimension of biodiversity that human activity affects and would serve as a basic survey for sustainable use of the sea.

Abstract Coral reefs represent some of the most biodiverse ecosystems in the world but are currently undergoing large‐scale degradation due to anthropogenic stressors. Such degradation usually begins with coral bleaching, and if the stress condition is inflicted for too long may eventually result in loss of structural complexity (or “flattening”) of the reef, dramatically changing habitat availability for reef‐associated fauna. Despite having been linked to important ecosystem functions, cryptobenthic organisms are often overlooked in ecological monitoring programs, and their microhabitat dependencies are poorly understood. Here, we combined collection‐based biodiversity monitoring techniques with five different environmental DNA (eDNA) sampling techniques (Reef water, sediment, crevice water, slurp gun, and bulk sediment) to survey cryptobenthic fishes and annelids on a Maldivian fringing coral reef. Collectively, 176 fish and 140 annelid taxa were detected with eDNA across 14 surveyed sites, more than doubling the reported annelid taxa in the region with 88 new occurrences. Water filtered near the reef structure revealed the highest species richness out of the five eDNA sampling techniques tested. Furthermore, we found correlations between fish species richness and topographic complexity for both collection‐ and eDNA‐based techniques. This suggests that detection by eDNA may be linked to site‐specific predictors and reveal community differences across small spatial scales (tens of meters). We also report that reef flattening (going from structural complex to less complex sites) can cause a 50% reduction in fish diversity and that cryptobenthic fish species richness was highly associated with branching corals. In contrast, annelid communities showed no clear correlations with environmental predictors, but co‐amplification of non‐target, non‐annelid taxa may have distorted such correlations if present. This suggest that the predictive powers of eDNA for environmental gradients may be dependent on the targeted taxa.

Abstract Aquatic fungi drive important ecosystem processes, which may be affected by anthropogenic stressors, including changes in water quality. However, due to the great diversity of aquatic fungi, our understanding of the relationships among water quality variables, fungal community composition, and ecosystem function remains incomplete. Here, we show that aquatic fungal community structure and functional diversity correlate strongly with water quality. We used a multi‐marker survey of environmental DNA and long‐term water quality data to survey 17 stream, pond, and tidal creek sites within a coastal drainage network in southeastern Virginia, USA, targeting both the ITS2 and LSU barcoding regions. The community composition of Chytridiomycota and Ascomycota (fungal phyla) and aquatic hyphomycetes (a functional group) were strongly correlated with habitat type and water quality variables. Functional diversity of aquatic fungi decreased with warmer water, due mainly to the reduced richness of saprotrophic groups, and increased with total particulate phosphorus, due to the richness of algal parasites. Community dissimilarity analyses of samples using both ITS2 and LSU yielded consistent results, but per‐sample alpha diversity differed. This work reveals a surprising degree of local variation in aquatic fungal community composition and functional diversity within a coastal watershed, which was coupled with variation in water quality variables. Furthermore, eDNA sampling is an effective and efficient means to accurately characterize diverse aquatic fungal communities with exciting implications for addressing basic and applied research questions. Our work supports the prediction that community composition and functional diversity of aquatic fungi are impacted by impaired water quality and global change.

Abstract The Northern muriqui (Brachyteles hypoxanthus) is one of the world's most critically endangered primates, with only ~1000 mature individuals remaining in the wild. Habitat loss and hunting have led to its sharp decline, making conservation efforts crucial. Analyses of gut microbiomes in wild populations can provide valuable information on host health and vulnerability, and ultimately, contribute to baseline knowledge toward improving conservation programs and reintroduction efforts. In this study, we analyzed the microbiome (16S rRNA metabarcoding) of fecal samples belonging to 53 uniquely genotyped individuals from three social groups from the Caparaó National Park, aiming to provide the first assessment of the microbiome diversity and composition for this species. Our results showed the muriqui gut microbiome was predominantly composed of the phyla Bacteroidetes and Firmicutes, with the dominant classes represented by Bacteroidia and Clostridia. High similarity in bacterial diversity and composition was found for individuals from distinct groups, suggesting a negligible geographical effect at the fine spatial scale analyzed. No significant effect of host genotype heterozygosity levels on microbiota diversity was recovered, but a significant influence of genetic distance on microbiota community structure and composition was demonstrated. Our findings stress the importance of considering associations between host genetics and the microbiome and suggest that the analyzed populations host a similar microbiome composition. This detailed microbiome assessment can aid conservation actions, including future anthropogenic impact assessments and animal reintroductions.