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DNA barcoding methods are used to identify organisms or groups of organisms based on DNA sequences within selected regions of a genome. These methods are useful in studying fish (and other aquatic organisms), as genetic material in the form of environmental DNA (eDNA) or cells is freely diffused in water. This allows researchers to identify what species are present in a body of water by collecting a water sample, extracting DNA from the sample and isolating DNA sequences that are specific for the species of interest[1]. Barcoding methods can also be used for biomonitoring and food safety validation, assessing food webs and species distribution, and for detection of invasive species[1].

In fish research, barcoding can be used as an alternative to more traditional sampling methods, and often they can provide more information without damage to the studied animals[2]. Barcoding methods can also be very cost efficient compared to the traditional methods [reference needed].

Aquatic environments have unique properties that affect how genetic material from organisms is distributed. DNA material diffuses rapidly in aquatic environment, which makes it possible to detect organisms from wide area when sampling from a specific spot[1]. Due to rapid degradation of DNA in aquatic environments, detected organism represent contemporary presence of these organisms, without confounding signals from the past[3].

One of the major benefits of DNA-based identifications is their fast, reliable and accurate characterization across all life stages and species[4]. Reference libraries are used to connect barcode sequences to single species and can be used to assess the organisms present in DNA samples. Libraries of reference sequences are also useful in identifying species in cases of morphological ambiguity, such as with larval stages[4].

Using eDNA samples and barcoding methods are important in water management, as the species composition of an aquatic environment can be used as an indicator of ecosystem health[5]. These methods are particularly useful in studying endangered or elusive fish, as species can be detected without catching or harming the animals[6].

Trachurus symmetricus baitball
Baitball of fish


Applications

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Ecological monitoring

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Biomonitoring of aquatic ecosystems is required by national and international legislation (e.g. WFD, MSFD). Traditional methods are time-consuming and include destructive practices that could injure individuals of protected species. DNA barcoding has been proposed as a relatively cheap and quick methods for identifying fish species in freshwater and marine environments. Presence or absence of key fish species can be detected using eDNA collected from water bodies. The distribution of species and their spatio-temporal patterns can be studied (e.g. spawning event timing and location). This can help discover the impacts of physical barriers (e.g. dam construction) and other human disturbances. DNA tools have been used in dietary studies of fish and the construction of aquatic food webs. Metabarcoding of the gut contents or feces of fish will identify the species they have recently consumed although secondary predation must be taken into consideration [7].

Common lion fish
Common lion fish

Invasive species

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Metabarcoding of eDNA (collected in water samples) can be used to detect cryptic or invasive species in aquatic ecosystems. Early detection is vital for the control and removal of these non-native and ecological harmful species (e.g. common dace (Leuciscus leuciscus) in Ireland or lion fish (Pteroissp.) in the Atlantic and Caribbean) [8].

Fisheries management

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Barcoding and meta-barcoding approaches yield rigorous and extensive data on recruitment, ecology and geographic ranges of fisheries resources and improves knowledge of nursery areas and spawning grounds, with evident benefits for fisheries management. Traditional methods for fishery assessment can often be highly destructive, such as electrofishing, gillnet sampling or trawling. Molecular methods offer non-invasive sampling methods. For example, barcoding and meta-barcoding 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. It is also a powerful tool in monitoring of fisheries quotas and by-catch[9].

Important for commercial fisheries is that eDNA can detect and quantify the abundance of some anadromous species as well as their temporal distribution which can be used to design the appropriate management measures.[10][11]

Food safety

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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[12][13]. A recent study from supermarkets in the state of New York discovered that 26.92% of seafood purchases with an identifiable barcode were mislabelled [14].

Barcoding as method can also trace fish species as human health hazard related to consumption of fish.  In some cases biotoxin can also be concentrated, when the toxin move up in food chain. One such example relates to coral reef species where predatory fish such as barracuda have been detected to cause (Ciguatera fish poisoning). Such new association of fish poisoning can be detected by the use of fish barcoding.

Confiscated shark fins

Protection of endangered species

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Barcoding can also be used in the conservation of endangered species through the prevention of illegal trading of CITES listed species. There is a significant black market for fish based foods and also in the aquarium and pet trades. To protect sharks from overexploitation, illegal use of these fishes can be found from barcoding shark fin soup and traditional medicines containing shark [15].

Barcoding parasites of fish

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When investigating fish or other aquatic animals it is likely that you will encounter other organism parasitizing these hosts externally, internally in muscle or visceral organs or hidden within certain cell types distributed within the host animals. In these cases, morphological identifications of the invading parasites depend on its visibility and external characteristics. If these parasites are detected often depends on the size, placement and visibility by the eye. Sometimes they might be hard to detect even by the use of low power microscopes. In these cases, and high resolution microscopical techniques and/or histological sections in combination with special staining must be used to find and hopefully correctly identify and place these parasites. Even so many metazoan parasites might not be identified down to species level and can only be placed in a higher taxonomical rank. This means that many parasites are missed or neglected when these investigations are performed leading to a great underestimation of the parasitic burden and their role in the ecosystem. In such cases barcoding could be a useful tool. Most metazoan parasites by the exception of Myxosporean parasites (Cnidarian group of organism) will be possible to place into different parasitic groups by use the same type of genetic markers that the BOLD system provides. However many times the use of DNAbarcode as a fingerprint might not lead all the way to species identification, and the resolution can be insufficient when comparing different strains of the same species, when other types of genetic markers are needed.

Methodology

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Sampling in aquatic environments

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Collecting eDNA samples

Aquatic environments have special attributes that would be considered in sampling for fish eDNA metabarcoding and it will vary according to that. Seawater sampling has a particular interest to the assessment of health of marine ecosystems and their biodiversity. Even though the dispersion of eDNA on seawater is larger than other water bodies and salinity influence negatively on DNA preservation, it contains high amounts of eDNA from fishes which can be detectable even 1 week after sampling. It was described that free molecules, intestinal scale and skin cell debris are the main resource of fish eDNA[16].

On the other hand, ponds have biological and chemical properties that could change eDNA recover and detection. The small size that pounds have comparing to other water bodies makes them more sentitive to natural conditions such as exposure to UV light, temperature and pH changes that affect the amount of eDNA. Moreover, trees and dense aquatic vegetation around ponds represent a real barrier that avoid aeration of water by the wind and could promote the accumulation of chemical species that could damage eDNA integrity [17]. Some studies have shown that heterogeneous distribution of eDNA in ponds may affect detection of fishes. Harper et.al. (2018) decribed that availability of fish eDNA is also dependent of life stage, activity, seasonality and behavior. The best amounts of eDNA are obtained from spawning, larval stages and breeding activity [18].

Target regions

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Primer design is crucial for metabarcoding success. Some studies focused on primer development have described cytochrome B and 16S as suitable target regions for fish metabarcoding experiments. Evans et.al. (2016) described that Ac16S and L2513/H2714 primer sets are able to detect fish species accurately in different mesocosms [19]. Another study performed by Valentini et.al. (2016) shown that L1848/H1913 primer pair, which amplifies a region of 12S rRNA locus, were able to reach high taxonomical coverage and discrimination even when the target was a short fragment. This research also evidenced that in 89% of sampling sites, metabarcoding approach was similar or even higher than traditional methods [20]. 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 target were located in mitochondrial 12S and cytochrome B regions respectively. Results demonstrate that detection of fish species were higher using 12S primers than CytB, this due to the longer persistence of shorter 12S fragment (~100bp) has in comparison with larger CytB amplicon (~460bp) [21]. In general, all these studies summarize that special considerations about primer design and selection has to be taken according to the objectives and nature of the experiment.

Fish reference databases

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There are a number of open access databases available to researchers worldwide. The proper identification of fish specimens with DNA barcoding methods relies heavily on the quality and species coverage of available sequence databases. A fish reference database is an electronic database that typically contains DNA barcodes, images, and geospatial coordinates of examined fish specimens. The database can also contain linkages to voucher specimens, information on species distributions, nomenclature, authoritative taxonomic information, collateral natural history information and literature citations.

FISH-BOL

Started in 2005, The Fish Barcode of Life Initiative (FISH-BOL) www.fishbol.org is an international research collaboration that is assembling a standardized reference DNA sequence library for all fishes[22]. It is a concerted global research project launched in 2005, with the goal to collect and assemble standardized DNA barcode sequences and associated voucher provenance data in a curated reference sequence library to aid the molecular identification of all fish species.[23]

If researchers wish to contribute to the FISH-BOL reference library, clear guidelines are provided for specimen collection, imaging, preservation, and archival, as well as meta-data collection and submission protocols[24]. The Fish-BOL database functions as a portal to the Barcode of Life Data Systems (BOLD).

A fish reference database is an electronic database that typically contains DNA barcodes, images, and geospatial coordinates of examined fish specimens. The database can also contain linkages to voucher specimens, information on species distributions, nomenclature, authoritative taxonomic information, collateral natural history information and literature citations.

French Polynesia Fish Barcoding Base

The French Polynesia Fish Barcoding Database contains all the captured specimens during several field trips organised by CRIOBE or participated since 2006 in Archipelagoes of French Polynesia. For each classified specimen, following information can be available: scientific name, picture, date, GPS point, depth and method of capture, size, and Cytochrome Oxidase c Subunit 1 (CO1) DNA sequence. The database can be searched using name (genus or species) or using a part of the CO1 DNA sequence.

Aquagene

A collaborative product developed by several German institutions, Aquagene provides free access to curated genetic information of marine fish species. Its searchable database allows species identification by DNA sequence comparisons. All species are characterized by multiple gene sequences, presently including the standard COI barcoding gene together with CYTB, MYH6 and (coming shortly) RHOD, facilitating unambiguous species determination even for closely related species or those with high intraspecific diversity. The genetic data is complemented online with additional data of the sampled specimen, such as digital images, voucher number and geographic origin.

Additional resources

Other reference database that are more general, but may also be useful for barcoding fish are the Barcode of Life Datasystem and Genbank. 

Reference databases may be curated, meaning that the entries are subjected to expert assessment before being included, or uncurated, in which case they may include a large number of reference sequences but with less reliable identification of species.

Advantages

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Barcoding/meta-barcoding provides quick and usually reliable species identification, and morphological identification, i.e. taxonomic expertise, it not needed. Meta-barcoding also makes it possible to identify species when organisms are degraded[25] or only a part of an organism is available. It is a powerful tool for detection of rare and/or invasive species, as can be detected despite low abundance in a water body. Traditional methods to assess fish biodiversity[6], abundance and density include the use of gears like nets, electrofishing equipment[6], trawls, cages, fyke-nets or other gear which show reliable results of presence only for abundant species. Contrary, rare native species, as well as newly established alien species, are less likely to be detected via traditional methods, leading to incorrect absence/presence assumptions[6]. Barcoding/meta-barcoding is also in some cases a non-invasive sampling method, as it provides the opportunity to analyze DNA from eDNA or by sampling living organisms[26][27][28].

For fish parasites, metabarcoding allows the detection of cryptic or microscopic parasites from aquatic environment, which is difficult with more direct methods. Some parasites exhibit cryptic variation and metabarcoding can be helpful method in revealing this[29].

The application of eDNA meta-barcoding is cost effective in large surveys or when many samples are required. eDNA can reduce the costs of fishing, transport of samples and time invested by taxonomists, and requires small amounts of DNA from target species to reach reliable detection in most cases. Constantly decreasing prices for barcoding/meta-barcoding due to technical development is another advantage[2][30][31].

Lastly, the eDNA approach is suitable for monitoring of inaccessible environments.

Challenges

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The results obtained from meta-barcoding are limited or biased to the frequency of occurrence. There is also the obvious challenge that far from all species have barcodes attached to them[25]. This challenge can partly be overcome by the use of OTU’s, although this is not a permanent solution. Further, contamination is a risk when using metabarcoding as a tool in ecological research, as such studies are rarely carried out under sterile conditions.

Even though meta-barcoding could solve practical limitations of conventional sampling methods, researchers have not provide consensus considerations of experimental design and bioinformatic criteria that has to be applied to define detection during eDNA metabarcoding. The lack of criteria is due to the influence of different factors such as fish diversity, fish abundancy, type of aquatic ecosystem, number of markers and marker specificity, which could be different depending on the experiment[31].

About meta-barcoding as tool in stock assessment, eDNA as the only source of information is not sufficient. That is because not all species can be barcoded, and because it provides no information about length, weight, sex or other characteristics of the detected species. It can, for now, thus only be seen as a complement to monitory fishing in e.g. ecological assessment.

Another significant challenge for the method is how to quantify fish abundance from molecular data. Although there are some cases in which quantification has been possible[32] there appears to be no consensus on how, or to what extent, molecular data can meet this aim for fish monitoring in general. Further refinements of the methods will likely lead to more accurate abundance estimates[33].

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International Barcode of Life: http://ibol.org

References

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  2. ^ a b Goldberg, Caren S.; Turner, Cameron R.; Deiner, Kristy; Klymus, Katy E.; Thomsen, Philip Francis; Murphy, Melanie A.; Spear, Stephen F.; McKee, Anna; Oyler-McCance, Sara J. (2016-11). Gilbert, M. (ed.). "Critical considerations for the application of environmental DNA methods to detect aquatic species". Methods in Ecology and Evolution. 7 (11): 1299–1307. doi:10.1111/2041-210X.12595. {{cite journal}}: Check date values in: |date= (help)
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  7. ^ Kim, Hyun-Woo; Park, Hyun; Baeck, Gun Wook; Lee, Jae-Bong; Lee, Soo Rin; Kang, Hye-Eun; Yoon, Tae-Ho (2017-11-07). "Metabarcoding analysis of the stomach contents of the Antarctic Toothfish (Dissostichus mawsoni) collected in the Antarctic Ocean". PeerJ. 5: e3977. doi:10.7717/peerj.3977. ISSN 2167-8359.{{cite journal}}: CS1 maint: unflagged free DOI (link)
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  12. ^ Barcaccia, Gianni; Lucchin, Margherita; Cassandro, Martino (2015-12-29). "DNA Barcoding as a Molecular Tool to Track Down Mislabeling and Food Piracy". Diversity. 8 (4): 2. doi:10.3390/d8010002. ISSN 1424-2818.{{cite journal}}: CS1 maint: unflagged free DOI (link)
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  18. ^ Harper, Lynsey R.; Buxton, Andrew S.; Rees, Helen C.; Bruce, Kat; Brys, Rein; Halfmaerten, David; Read, Daniel S.; Watson, Hayley V.; Sayer, Carl D. (2019-01-01). "Prospects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds". Hydrobiologia. 826 (1): 25–41. doi:10.1007/s10750-018-3750-5. ISSN 1573-5117.
  19. ^ Evans, Nathan T.; Olds, Brett P.; Renshaw, Mark A.; Turner, Cameron R.; Li, Yiyuan; Jerde, Christopher L.; Mahon, Andrew R.; Pfrender, Michael E.; Lamberti, Gary A. (2016-1). "Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding". Molecular Ecology Resources. 16 (1): 29–41. doi:10.1111/1755-0998.12433. PMC 4744776. PMID 26032773. {{cite journal}}: Check date values in: |date= (help)CS1 maint: PMC format (link)
  20. ^ Valentini, Alice; Taberlet, Pierre; Miaud, Claude; Civade, Raphaël; Herder, Jelger; Thomsen, Philip Francis; Bellemain, Eva; Besnard, Aurélien; Coissac, Eric (2016-2). "Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding". Molecular Ecology. 25 (4): 929–942. doi:10.1111/mec.13428. {{cite journal}}: Check date values in: |date= (help)
  21. ^ Hänfling, Bernd; Handley, Lori Lawson; Read, Daniel S.; Hahn, Christoph; Li, Jianlong; Nichols, Paul; Blackman, Rosetta C.; Oliver, Anna; Winfield, Ian J. (2016). "Environmental DNA metabarcoding of lake fish communities reflects long-term data from established survey methods". Molecular Ecology. 25 (13): 3101–3119. doi:10.1111/mec.13660. ISSN 1365-294X.
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  25. ^ a b Harms-Tuohy, Ca; Schizas, Nv; Appeldoorn, Rs (2016-10-25). "Use of DNA metabarcoding for stomach content analysis in the invasive lionfish Pterois volitans in Puerto Rico". Marine Ecology Progress Series. 558: 181–191. doi:10.3354/meps11738. ISSN 0171-8630.
  26. ^ Corse, Emmanuel; Costedoat, Caroline; Chappaz, Rã‰Mi; Pech, Nicolas; Martin, Jean-Franã‡Ois; Gilles, Andr㉠(2010-1). "A PCR-based method for diet analysis in freshwater organisms using 18S rDNA barcoding on faeces: DNA barcoding in diet of freshwater organisms". Molecular Ecology Resources. 10 (1): 96–108. doi:10.1111/j.1755-0998.2009.02795.x. {{cite journal}}: C1 control character in |first3= at position 3 (help); C1 control character in |first5= at position 11 (help); C1 control character in |first6= at position 6 (help); Check date values in: |date= (help)
  27. ^ Taguchi, T.; Miura, Y.; Krueger, D.; Sugiura, S. (2014-5). "Utilizing stomach content and faecal DNA analysis techniques to assess the feeding behaviour of largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus: stomach content and faecal dna analysis". Journal of Fish Biology. 84 (5): 1271–1288. doi:10.1111/jfb.12341. {{cite journal}}: Check date values in: |date= (help)
  28. ^ Guillerault, N.; Bouletreau, S.; Iribar, A.; Valentini, A.; Santoul, F. (2017-5). "Application of DNA metabarcoding on faeces to identify European catfish Silurus glanis diet: dna metabarcoding of s. glanis faeces". Journal of Fish Biology. 90 (5): 2214–2219. doi:10.1111/jfb.13294. {{cite journal}}: Check date values in: |date= (help)
  29. ^ Hartikainen, Hanna; Gruhl, Alexander; Okamura, Beth (2014-7). "Diversification and repeated morphological transitions in endoparasitic cnidarians (Myxozoa: Malacosporea)". Molecular Phylogenetics and Evolution. 76: 261–269. doi:10.1016/j.ympev.2014.03.010. {{cite journal}}: Check date values in: |date= (help)
  30. ^ Valentini, Alice; Taberlet, Pierre; Miaud, Claude; Civade, Raphaël; Herder, Jelger; Thomsen, Philip Francis; Bellemain, Eva; Besnard, Aurélien; Coissac, Eric (2016-2). "Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding". Molecular Ecology. 25 (4): 929–942. doi:10.1111/mec.13428. {{cite journal}}: Check date values in: |date= (help)
  31. ^ a b Evans, Nathan T.; Li, Yiyuan; Renshaw, Mark A.; Olds, Brett P.; Deiner, Kristy; Turner, Cameron R.; Jerde, Christopher L.; Lodge, David M.; Lamberti, Gary A. (2017-9). "Fish community assessment with eDNA metabarcoding: effects of sampling design and bioinformatic filtering". Canadian Journal of Fisheries and Aquatic Sciences. 74 (9): 1362–1374. doi:10.1139/cjfas-2016-0306. ISSN 0706-652X. {{cite journal}}: Check date values in: |date= (help)
  32. ^ Maruyama, Atsushi; Sugatani, Kousuke; Watanabe, Kazuki; Yamanaka, Hiroki; Imamura, Akio (2018). "Environmental DNA analysis as a non-invasive quantitative tool for reproductive migration of a threatened endemic fish in rivers". Ecology and Evolution. 8 (23): 11964–11974. doi:10.1002/ece3.4653. PMC 6303803. PMID 30598791.{{cite journal}}: CS1 maint: PMC format (link)
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