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Rivers are a vital resource for human wellbeing. To reduce human impact on water bodies, the European Union has established an essential regulatory framework for protection and sustainable management (WFD; 2000/60/EC). In this strategy, reliable and economic bioindicators are a fundamental component. Benthic macroinvertebrates are the group most commonly used as bioindicators through all European countries. However, their conventional assessment currently entails serious cost-efficiency limitations. In this study, we have tested the reliability of metabarcoding as a tool to record river macroinvertebrates using samples from a mock community (in vitro validation) and eDNA extracted for field validation from water from six sites within a north Iberian river (River Nalón, Asturias, Spain). Two markers (V4 region within the nuclear 18S rDNA and a fragment of the mitochondrial COI gene) were amplified and sequenced using an Illumina platform. The molecular technique has proven to be more sensitive than the visual one. A cost-benefit analysis shows that the metabarcoding approach is more expensive than conventional techniques for determining macroinvertebrate communities but requires fewer sampling and identification efforts. Our results suggest metabarcoding is a useful tool for alternative assessment of freshwater quality.

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BackgroundSalmonids are native from the North Hemisphere but have been introduced for aquaculture and sport fishing in the South Hemisphere and inhabit most rivers and lakes in temperate and cold regions worldwide. Five species are included in the Global Invasive Species Database: rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar, brown trout Salmo trutta, brook trout Salvelinus fontinalis, and lake trout Salvelinus namaycush. In contrast, other salmonids are endangered in their native settings.MethodsHere we have developed a method to identify salmonid species directly from water samples, focusing on the Iberian Peninsula as a case study. We have designed nested Salmonidae-specific primers within the 16S rDNA region. From these primers and a PCR-RFLP procedure the target species can be unequivocally identified from DNA extracted from water samples.ResultsThe method was validated in aquarium experiments and in the field with water from watersheds with known salmonid populations. Finally, the method was applied to obtain a global view of the Salmonidae community in Nalón River (north coast of Spain).DiscussionThis new powerful, very sensitive (identifying the species down to 10 pg DNA/ml water) and economical tool can be applied for monitoring the presence of salmonids in a variety of situations, from checking upstream colonization after removal of river barriers to monitoring potential escapes from fish farms.

Background: Bioassessment of rivers is a fundamental method to determine surface water quality. One of the groups most commonly employed as bioindicators of aquatic ecosystems are benthic macroinvertebrates. Their conventional assessment is based on morphological identification and entails several limitations, such as being time-consuming and requires trained experts for taxonomic identification. The use of genetic tools to solve these limitations offers an alternative way to evaluate rivers status. The use of environmental DNA (eDNA) metabarcoding has increased in recent years for different purposes, but its use in water quality evaluation is yet to be tested. Here, morphological and eDNA based inventories of macroinvertebrates were compared from the same seven sampling sites in the Upper Nalón River Basin (Asturias, Spain). Materials & Methods:High-Throughput Sequencing (HTS) of the cytochrome oxidase subunit 1 (COI) gene was carried out on DNA from water samples using an Ion Torrent platform. Biotic water quality indices were calculated from morphological and molecular data and compared with independent physico-chemical habitat assessment to validate eDNA based approach.Results: Highly positive and significant correlation was found between eDNA metabarcoding and morphological methods (Morphological and eDNA indices, r = 0.798, 5 degrees of freedom d.f., P = 0.031;) and a highly significant negative correlation was found between molecular and habitat quality indices (Stress score & eDNA, ρ = -0.878 and P = 0.009; Stress score & Visual, ρ = -0.949 and P = 0.0002). Discussion:The similarity of results from the two approaches and the correlation of eDNA metabarcoding data with the habitat quality indices, suggest that eDNA performs as well as conventional methods for calculating biotic indices in this system, positioning eDNA metabarcoding of macroinvertebrate communities to transform how river bioassessment can be achieved.

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Biological invasions have increased in recent decades due to globalization and human activities. These invasions are currently one of the main threats to biodiversity, and their early detection is essential for a rapid and effective response. Here, we explored the use of citizen science strategies to create an early alert to detect invasive species. Our main objective was to evaluate the general knowledge of volunteer participants of invasive freshwater species in Asturias (north of the Iberian Peninsula) and compare it with both real data from electrofishing surveys and official data from the regional government. A total of 140 volunteer surveys were conducted in four different rivers in Asturias. The largest group of participants consisted of males older than 50 years. Four species were identified as native to the four rivers: Anguilla anguilla; Mugil cephalus; Salmo salar; and, Salmo trutta. More than 50% of the native species surveyed by electrofishing were recognized by the locals in each river region. A total of 22.86% of the volunteers were able to correctly name an exotic species, and a total of 7 were correctly identified: Procambarus clarkii; Trachemys scripta; Cyprinus carpio; Esox lucius; Salvelinus fontinalis; Carassius auratus; and, Oncorhynchus mykiss. However, compared to the list of actual exotic species surveyed, less than 40% were recognized in the four rivers. Despite the poor correlation between local knowledge and real exotic aquatic fauna, citizens were able to detect one exotic species not yet found in the wild in this region (T. scripta). Finally, more than 70% of the volunteers were in favor of fighting against invasive species, although only 22.86% were able to identify any specific exotic species found in the region. The positive attitude to exotic species control was correlated with both the level of native species knowledge and the concern about the ability of exotic species to impact native fauna in the region. Better training will improve public awareness, reduce the nonintentional release of non-native species, and increase the detection of non-indigenous species. The attitudes of the citizens make the region a promising candidate for education efforts to reduce alien species introductions and help preserve fauna biodiversity.

BackgroundMany fish species have been introduced in wild ecosystems around the world to provide food or leisure, deliberately or from farm escapes. Some of those introductions have had large ecological effects. The north American native rainbow trout (Oncorhynchus mykiss Walbaum, 1792) is one of the most widely farmed fish species in the world. It was first introduced in Spain in the late 19th century for sport fishing (Elvira 1995) and nowadays is used there for both fishing and aquaculture. On the other hand, the European native brown trout (Salmo trutta L.) is catalogued as vulnerable in Spain. Detecting native and invasive fish populations in ecosystem monitoring is crucial, but it may be difficult from conventional sampling methods such as electrofishing. These techniques encompass some mortality, thus are not adequate for some ecosystems as the case of protected areas. Environmental DNA (eDNA) analysis is a sensitive and non-invasive method that can be especially useful for rare and low-density species detection and inventory in water bodies.MethodsIn this study we employed two eDNA based methods (qPCR and nested PCR-RFLP) to detect salmonid species from mountain streams within a protected area, The Biosphere Reserve and Natural Park of Redes (Upper Nalón Basin, Asturias, Northern Spain), where brown trout is the only native salmonid. We also measured some habitat variables to see how appropriate for salmonids the area is. The sampling area is located upstream impassable dams and contains one rainbow trout fish farm.ResultsEmploying qPCR methodology, brown trout eDNA was detected in all the nine sampling sites surveyed, while nested PCR-RFLP method failed to detect it in two sampling points. Rainbow trout eDNA was detected with both techniques at all sites in the Nalón River’ (n1, n2 and n3). Salmonid habitat units and water quality were high from the area studied.DiscussionIn this study, a high quantity of rainbow trout eDNA was found upstream and downstream of a fish farm located inside a Biosphere Reserve. Unreported escapes from the fish farm are a likely explanation of these results. Since salmonid habitat is abundant and the water quality high, the establishment of rainbow trout populations would be favored should escapes occur. Environmental DNA has here proved to be a valuable tool for species detection in freshwater environments, and the probe-based qPCR highly sensitive technique for detection of scarce species. We would recommend this method for routine monitoring and early detection of introduced species within natural reserves.