Improving the detection of rare native fish species in environmental DNA metabarcoding surveys

The presence of threatened or endangered species often strongly influences management and conservation decisions. Within the Murray–Darling Basin (MDB), Australia, the presence of threatened native fish affects the management and allocation of water resources. These decisions are currently based on traditional fisheries data and a predictive MaxEnt model. However, it is important to verify the model's predictive power given the implication it may have, but this requires methods with a high detection sensitivity for rare species. Although the use of environmental DNA (eDNA) metabarcoding achieves a higher detection sensitivity compared with traditional methods, earlier surveys in the MDB have shown that the highly abundant and invasive common carp (Cyprinus carpio) can reduce detection probabilities for rare species. Consequently, a polymerase chain reaction (PCR) blocking primer designed to block the amplification of carp eDNA could increase the detection probabilities for rare native species while simultaneously reducing the required sampling effort and survey costs. Although PCR blocking primers are often used in ancient DNA and dietary studies, no aquatic eDNA metabarcoding study to date has evaluated the potential benefits of using PCR blocking primers. A laboratory and field‐based pilot study was used to address this knowledge gap and assess the impact of a blocking primer on the detection probabilities of native species and the minimum sampling effort required. Results showed that the inclusion of the blocking primer increased the detection probabilities for native species by 10–20% and reduced the minimum required sampling effort by 25–50%. These findings provide important insights into possible methods for optimizing eDNA metabarcoding surveys for the detection of rare aquatic species

Growth of brown trout in the wild predicted by embryo stress reaction in the laboratory

Laboratory studies on embryos of salmonids, such as the brown trout (Salmo trutta), have been extensively used to study environmental stress and how responses vary within and between natural populations. These studies are based on the implicit assumption that early life-history traits are relevant for stress tolerance in the wild. Here we test this assumption by combining two datasets from studies on the same 60 full-sib families. These families had been experimentally produced from wild breeders to determine, in separate samples, (i) stress tolerances of singly kept embryos in the laboratory and (ii) growth of juveniles during 6 months in the wild. We found that growth in the wild was well predicted by larval size of their full sibs in the laboratory, especially if these siblings had been experimentally exposed to a pathogen. Exposure to the pathogen had not caused elevated mortality among the embryos but induced early hatching. The strength of this stress-induced change of life history was a significant predictor of juvenile growth in the wild: the stronger the response in the laboratory, the slower the growth in the wild. We conclude that embryo performance in controlled environments can be useful predictors of juvenile performance in the wild

Monitoring riverine fish communities through eDNA metabarcoding:Determining optimal sampling strategies along an altitudinal and biodiversity gradient

Monitoring aquatic biodiversity through DNA extracted from environmental samples (eDNA) combined with high-throughput sequencing, commonly referred to as eDNA metabarcoding, is increasing in popularity within the scientific community. However, sampling strategies, laboratory protocols and analytical pipelines can influence the results of eDNA metabarcoding surveys. While the impact of laboratory protocols and analytical pipelines have been extensively studied, the importance of sampling strategies on eDNA metabarcoding surveys has not received the same attention. To avoid underestimating local biodiversity, adequate sampling strategies (i.e. sampling intensity and spatial sampling replication) need to be implemented. This study evaluated the impact of sampling strategies along an altitudinal and biodiversity gradient in the upper section of the Murrumbidgee River (Murray-Darling Basin, Australia). An eDNA metabarcoding survey was used to determine the local fish biodiversity and evaluate the influence of sampling intensity and spatial sampling replication on the biodiversity estimates. The results show that optimal eDNA sampling strategies varied between sites and indicate that river morphology, species richness and species abundance affect the optimal sampling intensity and spatial sampling replication needed to accurately assess the fish biodiversity. While the generality of the patterns will need to be confirmed through future studies, these findings provide a basis to guide future eDNA metabarcoding surveys in river systems

Evaluating genetic traceability methods for captive bred marine fish and their applications in fisheries management and wildlife forensics

Growing demands for marine fish products is leading to increased pressure on already depleted wild populations and a rise in the aquaculture production. Consequently, more captive bred fish are released into the wild through accidental escape or deliberate restocking, stock enhancement and sea ranching programs. The increased mixing of captive bred fish with wild conspecifics may affect the ecological and/or genetic integrity of wild fish populations. From a fisheries management perspective unambiguous identification tools for captive bred fish will be highly valuable to manage risks. Additionally there is great potential to use these tools in wildlife forensics (i.e. tracing back escapees to their origin and determining mislabelling of seafood products). Using SNP data from captive bred and wild populations of Atlantic cod (Gadus morhua L.) and sole (Solea solea L.), we explored the efficiency of population and parentage assignment techniques for the identification and tracing of captive bred fish. Simulated and empirical data were used to correct for stochastic genetic effects. Overall, parentage assignment performed well when a large effective population size characterizes the broodstock and escapees originate from early generations of captive breeding. Consequently, parentage assignments are particularly useful from a fisheries management perspective to monitor the effects of deliberate releases of captive bred fish on wild populations. Population assignment proved to be more efficient after several generations of captive breeding, which makes it a useful method in forensic applications for well-established aquaculture species. We suggest the implementation of a case by case strategy when choosing the best method

Alpine freshwater fish biodiversity assessment: an inter-calibration test for metabarcoding method set up

The analysis of environmental DNA (eDNA) by high throughput sequencing (HTS) is proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework Directive (WFD, 2000/60/EC), especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. These new biomonitoring methods based on eDNA show several advantages compared to classical morphological methods. The sampling procedures are easier and cheaper and eDNA metabarcoding is non-invasive and very sensitive, allowing for the detection of traces of DNA. However, eDNA metabarcoding methods need careful standardization to make the results of different surveys comparable. The aim of the EU project Eco-AlpsWater is to test and validate molecular biodiversity monitoring tools for aquatic ecosystems (i.e., eDNA metabarcoding) to improve the traditional WFD monitoring approaches in Alpine waterbodies. To this end, an inter-calibration test was performed using fish mock community samples containing either tissue-extracted DNA, eDNA collected from aquaculture tanks and eDNA samples collected from Lake Bourget (France). Samples were analysed using a DNA metabarcoding approach, relying on the amplification and HTS of a 12S rDNA marker, in two separate laboratories, to evaluate if different laboratory and bioinformatic protocols can provide a reliable and comparable description of the fish communities in both mock and natural samples. Our results highlight good replicability of the molecular laboratory protocols for HTS and good amplification success of selected primers, providing essential information concerning the taxonomic resolution of the 12S mitochondrial marker in describing the Alpine fish communities. Interestingly, different concentrations of species DNA in the mock samples were well represented by the relative DNA reads abundance. These tests confirm the reproducibility of eDNA metabarcoding analyses for the biomonitoring of freshwater fish inhabiting Alpine and peri-Alpine lakes and river

Sex-Specific Life History Affected by Stocking in Juvenile Brown Trout

Salmonids are a socioeconomically and ecologically important group of fish that are often managed by stocking. Little is known about potential sex-specific effects of stocking, but recent studies found that the sexes differ in their stress tolerances already at late embryonic stage, i.e., before hatchery-born larvae are released into the wild and long before morphological gonad formation. It has also been speculated that sex-specific life histories can affect juvenile growth and mortality, and that a resulting sex-biassed demography can reduce population growth. Here we test whether juvenile brown trout (Salmo trutta) show sex-specific life histories and whether such sex effects differ in hatchery- and wild-born fish. We modified a genetic sexing protocol to reduce false assignment rates and used it to study the timing of sex differentiation in a laboratory setting, and in a large-scale field experiment to study growth and mortality of hatchery- and wild-born fish in different environments. We found no sex-specific mortality in any of the environments we studied. However, females started sex differentiation earlier than males, and while growth rates were similar in the laboratory, they differed significantly in the field depending on location and origin of fish. Overall, hatchery-born males grew larger than hatchery-born females while wild-born fish showed the reverse pattern. Whether males or females grew larger was location-specific. We conclude that juvenile brown trout show sex-specific growth that is affected by stocking and by other environmental factors that remain to be identified

Data from: An environmental DNA-based method for monitoring spawning activity: a case study, using the endangered Macquarie perch (Macquaria australasica)

Determining the timing and location of reproductive events is critical for efficient management of species. However, methods currently used for aquatic species are costly, time intensive, biased and often require destructive or injurious sampling. Hence, developing a non-invasive sampling method to accurately determine the timing and location of reproduction for aquatic species would be extremely valuable. We conducted an experimental and field study to determine the influence of spawning, and the mass release of spermatozoa in particular, on environmental DNA (eDNA) concentrations. Using a quantitative PCR approach we monitored changes in nuclear and mitochondrial eDNA concentrations over time. The data from the experimental study and the field survey supported our hypothesis that spawning events are characterized by higher concentrations of nuclear relative to mitochondrial eDNA. Outside of the reproductive period, we find that nuclear and mitochondrial DNA fragments are equally abundant in environmental water samples. We have shown that changes in the relative abundance of nuclear and mitochondrial eDNA can be used to monitor spawning activity of the endangered Macquarie perch. Our method is likely to be transferrable to other aquatic species and can be particularly useful to increase our understanding of the spawning biology of cryptic, rare or threatened species as well as design and evaluate environmental management actions and determine species establishment