Fish DNA barcoding

Biomonitoring of aquatic ecosystems is required by national and international legislation (e.g. the Water Framework Directive and the Marine Strategy Framework Directive). Traditional methods are time-consuming and include destructive practices that can harm individuals of rare or protected species. DNA barcoding is a relatively cost-effective and quick method for identifying fish species aquatic environments.[7] Presence or absence of key fish species can be established using eDNA from water samples and spatio-temporal distribution of fish species (e.g. timing and location of spawning) can be studied.[8] This can help discover e.g. impacts of physical barriers such as dam construction and other human disturbances. DNA tools are also used in dietary studies of fish and the construction of aquatic food webs. Metabarcoding of fish gut contents or feces identify recently consumed prey species. However, secondary predation must be taken into consideration.[9]

Early detection is vital for control and removal of non-indigenous, ecologically harmful species (e.g. lion fish (Pteroissp.) in the Atlantic and Caribbean). Metabarcoding of eDNA can be used to detect cryptic or invasive species in aquatic ecosystems.[10]

Barcoding and metabarcoding approaches yield rigorous and extensive data on recruitment, ecology and geographic ranges of fisheries resources. The methods also improve knowledge of nursery areas and spawning grounds, with benefits for fisheries management. Traditional methods for fishery assessment can be highly destructive, such as gillnet sampling or trawling. Molecular methods offers an alternative for non-invasive sampling. For example, barcoding and metabarcoding can help identifying fish eggs to species to ensure reliable data for stock assessment, as it has proven more reliable than identification via phenotypic characters. Barcoding and metabarcoding are also powerful tools in monitoring of fisheries quotas and by-catch.[11]

eDNA can detect and quantify the abundance of some anadromous species as well as their temporal distribution. This approach can be used to develop appropriate management measures, of particular importance for commercial fisheries.[12][13]

Globalisation of food supply chains has led to an increased uncertainty of the origin and safety of fish-based products. Barcoding can be used to validate the labelling of products and to trace their origin. “Fish fraud” has been discovered across the globe.[14][15] A recent study from supermarkets in the state of New York found that 26.92% of seafood purchases with an identifiable barcode were mislabelled.[16]

Barcoding can also trace fish species as there can be human health hazards related to consumption of fish. Further, biotoxins can occasionally be concentrated when toxins move up the food chain. One example relates to coral reef species where predatory fish such as barracuda have been detected to cause Ciguatera fish poisoning. Such new associations of fish poisoning can be detected by the use of fish barcoding.

Confiscated shark fins

Barcoding can be used in the conservation of endangered species through the prevention of illegal trading of CITES listed species. There is a large black market for fish based products and also in the aquarium and pet trades. To protect sharks from overexploitation, illegal use can be detected from barcoding shark fin soup and traditional medicines.[17]

Collecting eDNA samples

Aquatic environments have special attributes that need to be considered when sampling for fish eDNA metabarcoding. Seawater sampling is of particular interest for assessment of health of marine ecosystems and their biodiversity. Although the dispersion of eDNA in seawater is large and salinity negatively influences DNA preservation, a water sample can contain high amounts of eDNA from fish up to one week after sampling. Free molecules, intestinal lining and skin cell debris are the main sources of fish eDNA.[18]

In comparison to marine environments, ponds have biological and chemical properties that can alter eDNA detection. The small size of ponds compared to other water bodies makes them more sensitive to environmental conditions such as exposure to UV light and changes in temperature and pH. These factors can affect the amount of eDNA. Moreover, trees and dense vegetation around ponds represent a barrier that prevents water aeration by wind. Such barriers can also promote the accumulation of chemical substances that damage eDNA integrity.[19] Heterogeneous distribution of eDNA in ponds may affect detection of fishes. Availability of fish eDNA is also dependent of life stage, activity, seasonality and behavior. The largest amounts of eDNA are obtained from spawning, larval stages and breeding activity.[20]

Primer design is crucial for metabarcoding success. Some studies on primer development have described cytochrome B and 16S as suitable target regions for fish metabarcoding. Evans et.al. (2016) described that Ac16S and L2513/H2714 primer sets are able to detect fish species accurately in different mesocosms.[21] Another study performed by Valentini et.al. (2016) showed that the L1848/H1913 primer pair, which amplifies a region of 12S rRNA locus, was able to reach high taxonomical coverage and discrimination even with a short target fragment. This research also evidenced that in 89% of sampling sites, metabarcoding approach was similar or even higher than traditional methods (e.g. electrofishing and netting methods).[22] Hänfling et.al. (2016) performed metabarcoding experiments focused on lake fish communities using 12S_F1/12S_R1 and CytB_L14841/CytB_H15149 primer pairs, whose targets were located in the mitochondrial 12S and cytochrome B regions respectively. The results demonstrate that detection of fish species was higher when using 12S primers than CytB. This was due to the persistence of shorter 12S fragments (~100 bp) in comparison to larger CytB amplicon (~460 bp).[23] In general, these studies summarize that special considerations about primer design and selection have to be taken according to the objectives and nature of the experiment.