Precise determination of fish species and their origin

Every living creature, and thus every species of fish and aquatic animal, has a natural “signature” which is encoded in its genetic material. The sequence of molecular building blocks (nucleotides) in the genetic molecule, the DNA, is unique for every individual. Being able to read this nucleotide sequence enables conclusions to be drawn about the species in question – not only in the case of raw fishes but often also of cooked, smoked, marinated or sterilised products. 

 It was allegedly by chance that Alec Jeffreys on 10 September 1984 discovered a phenomenon that is today known as “genetic fingerprinting“. The British geneticist was at that time working on “minisatellites”, or tiny areas in the human DNA that stand out for their extremely high variability. The images of minisatellites from blood samples taken from members of one family could on the one hand be assigned to each family member and so were – like a fingerprint – individually specific for every single person. On the other hand, however, they also had some characteristics in common which made it possible to see family relationships between the individual persons. Jeffreys immediately recognised the practical significance of his discovery for with this tool it is possible to assign even a hair or skin particle with high accuracy to a person, or prove (or disprove) paternity.

As in all organisms the genetic information of fish and seafood is encoded in nucleic acids. This genetic code constitutes the blueprint and the rules for nearly all biological processes in a living organism. During fertilisation when the sperm cell fuses with the nucleus of the female egg cell it is newly combined and varied, and prior to every cell division duplicated and passed on to both daughter cells. About 99% of genetic information is concentrated in the chromosomal DNA of the cell nucleus; the remainder is in the mitochondria. These are tiny cell organelles in whose membranes important physiological processes such as the breakdown of fatty acids, parts of the urea cycle, but most importantly ATP (Adenosine Triphosphate) synthase are located. ATP is the “fuel” behind a lot of life processes which is why the mitochondria are also called the cells’ “power plants”. Mitochondria are only passed on to offspring by the mother (maternal inheritance) and new mitochondria can only be produced by the division of existing mitochondria. These properties make the usually circular DNA contained in the mitochondria (mtDNA) – which accounts for less than 1% of the cell’s DNA – particularly interesting for genetic analyses.

The DNA molecule is a double helical structure consisting of two parallel spiral strands in which nucleotides arranged complementarily to one another are connected rather like a rope-ladder. The arrangement or order of the nucleotides in the one strand (sequence) therefore determines the sequence of the other strand so that the strands of the double helix fit together like the teeth of a zip. This principle forms the basis for distinction between the different species: the more exactly the individual strands of the DNA molecule fit together (hybridise) the more closely they are related to one another. The DNA sequences of non-related living organisms hybridise less strongly or not at all. In the meantime DNA analysis has advanced a considerable step further for with today’s inventory of methods DNA sequences can be cut up and reassembled. Particularly interesting sections can be specifically removed and reproduced in millions of copies. Using modern sequencing machines it is today possible to decode the order of the nucleotides in the DNA molecule strand in a relatively short time. These methods have revolutionised biology and enable insights that would have been totally inconceivable just a few years ago. This gives rise to considerable expectations – some of which will probably only be able to be fulfilled in the more distant future, however. DNA sequencing of genetic material offers the unique chance, for example, to explore the causes of genetically induced diseases and to combat them effectively.

With DNA sequencing the processing state of the product is not important. Species classification is possible even with smoked eel.

Counterfeiters can be tracked down faster

Considerable progress has been made with regard to differentiation of various species. Within the species it is even possible to distinguish between very precise individuals. A decisive advantage of DNA analysis is its very high individual-specific significance. Today, if a DNA test has been performed correctly hardly anyone will doubt the results because this method has achieved worldwide recognition. Another advantage is that only extremely small amounts of the sample are necessary for DNA analyses… often just a drop of blood, a few cells from the body tissue, or a scale is sufficient.

However, this only works because suitable sequences from the DNA strand can be cut out and reproduced millions of times using a special technique called Polymerase Chain Reaction (PCR) in order to have enough material for the subsequent analyses. The analysis of the PCR products can be done in various ways using different methods. One is the sequencing of the nucleotide sequence in the DNA strand that can subsequently be compared with other sequences in a gene database to identify the appropriate fish species. Another method of analysis is called Single Strand Conformation Polymorphism (SSCP). Here several techniques are combined with one another. After the DNA fragments have been amplified with the help of PCR they are dotted onto a gel and subjected to an electric field. Due to their different electric charges the fragments move at different rates which leads to characteristic DNA banding patterns that can be made visible using special dyes. Often, the replacement of a single nucleotide in a strand with 100 bases can lead to an altered DNA banding pattern.

The network of certified test labs that can carry out such analyses has grown considerably in recent years and the costs of DNA analyses have fallen noticeably. Despite this, the question remains of course as to when such efforts and expense are useful and helpful, or what we can actually gain from such information, i.e. when a company should make use of this possibility. In brief it can be said to be useful when there is any doubt about whether a delivered product is exactly what was ordered, or whether it is worth the money paid. The variety of fish species and seafood that is traded on the global market has increased dramatically and the product in the pack is not always what was promised on the label. Minor and major frauds are to be found especially where expensive fish species and seafood products are concerned, particularly since these are often imported and are largely unknown elsewhere. And because these very popular fish species are often overfished the temptation is high to replace scarce supply with cheaper products. It is a fact that the control authorities are increasingly discovering falsely declared goods, particularly fillets that cannot be assigned to an individual fish species at a glance. At the top of this list of frauds is sole, one of the most expensive and popular fishes: some customers were in the past foisted off with tropical sole, witch or even pangasius fillets. Buyers are also sometimes deceived in deliveries of anglerfish, red snapper or some tuna species. Frauds of this kind could be proved undoubtedly using DNA analysis, the species identified, and the deception prevented.

Two out of four sushi restaurants and six out of ten fish shops that were investigated in New York had made false statements about the species they used.

In 2009, for example, a number of fish stores and sushi restaurants in New York were investigated: samples of the used fish were taken and subjected to genetic analysis. When the resulting DNA profiles were compared with information from a gene bank it became apparent that two of the four restaurants and six of the ten stores had given false information. The snappers and white tuna were in fact completely different fish species. DNA analyses are even more important, however, in the case of fish products that are subject o international trade restrictions such as sturgeon caviar. Without the instrument of DNA analysis which can be used in cases of doubt it would presumably be impossible for the customs authorities to control adherence to the CITES regulations. Even experts have difficulty identifying all caviar types on optical appearance only, particularly since the product range is today not only limited to Beluga, Osietra or Sevruga but there are also caviar from numerous other sturgeon species circulating in the markets. With the help of DNA analysis it can be clearly verified from which sturgeon species the caviar comes and whether it was caught in the wild or was produced in aquaculture.

 

DNA sequencing enables traceability right back to the fish stock

With DNA analysis it does not usually matter in which processing state a product is. For example, it is possible to determine an eel species in both raw and smoked condition. Using PCR and SSCP, suspicious products can be tested quickly to find out whether the eels hanging in the smoker are Anguilla anguilla or A. japonica or perhaps A. rostrata, A. bicolor or A. australis. DNA analyses also offer reliable results for canned products. In June 2010 Greenpeace published the results of a study which the Spanish molecular lab ATZI-Tecnalia had carried out on behalf of the environmentalists. The study of 165 cans of tuna from 50 different brands showed that in 30% of the examined cases the contents of the cans did not match the information on the labels. Some of the disputed cans contained completely different tuna species than marked and in some cans the different tuna species were mixed.

To ensure that they do not sell their customers low-quality fish for a higher price some companies have already taken DNA analyses into their routine screening programmes. The German frozen foods supplier Eismann has high-value species such as sole, turbot or John Dory tested in cases of doubt by specialised laboratories using DNA analysis. State authorities also make use of this possibility to track down counterfeiters and forgers. The US food agency FDA is currently in the process of installing a national DNA fish testing programme. DNA tests are also already under discussion as an additional means of ensuring certification programmes such as MSC. This seems to be necessary for researchers at Clemson University have with the help of DNA analyses determined that some of the fish that come onto the market with the MSC label that are not from sustainable fishing. The study had looked at Patagonian toothfish. The only fishery for this species that has so far been certified by the Marine Stewardship Council operates off the sub-Antarctic island of South Georgia. DNA analysis revealed that 8% of the traded fishes were not Patagonian toothfish at all and 15% of the genuine Patagonian toothfishes came from other not yet certified stocks. MSC is now increasingly using DNA analyses in order to ensure the traceability of the fish from the counter right back to the catch.

The EU Commission, too, plans to use DNA analysis for examining fish and fishery products in the future. The Joint Research Centre of the EU Commission presented analytical possibilities in May 2011 with which fish species and their origin can be determined more accurately than up to now. Already now it is clear that the tests will not only be limited to better monitoring and controls within the import and export business. The modern scientific methods can also contribute towards combating illegal fishing. In the project FishPopTrace which the EU is financing with 3.9 m EUR an international team of researchers is already investigating new tests with which the origin of a fish can be traced right back to the original stock and marine region. The results so far have been very encouraging. The most promising test uses single nucleotide polymorphisms, or SNP for short. These are certain mutations in the genetic make-up which occur in all living creatures and, among other things, depend on where the stocks of a species live. The FishPopTrace team has analysed the DNA of four important fish species of the European fishing sector (herring, cod, sole, and European hake) to find out which SNP is characteristic of the stocks in the different regions. With only 20 SNPs they were able to distinguish reliably between cod from the Atlantic and cod from the Baltic. One SNP alone was sufficient to distinguish sole from the North Sea from sole from the Mediterranean. And differentiation was also possible for herring and hake.

There is still some way to go, however, for the methods and data should be so precise that they could serve as evidence in court cases. Strangely, it is the very accuracy of the method that is causing concern among critics. On the one hand it is very good for proving false labelling of products by criminal traders. On the other hand it could lead to false suspicions because it cannot be fully ruled out that fishes sometimes end up in the fishermen’s nets whose origin is not clear to them. No one can rule out, for example, that the various stocks do not mix at the boundaries of their natural range.

 

More and more DNA profiles of fish species being stored in data bases

One essential prerequisite for the use of DNA analyses are data bases in which a maximum number of DNA profiles including in particular the profiles of common commercial fish and seafood species is stored. This is necessary in order to be able to compare the found nucleotide sequences with other profiles for species identification. The DNA profiles of as many fish species as possible are collected, for example as part of the big Fish Barcode of Life Project in Guelph, Ontario. The GenBank of the National Center of Biotechnology NCBI (http://www.ncbi.nlm.nih.gov/) also has a considerable date collection. In the EU the “Fishtrace” data base (www.fishtrace.org) is available to analysis labs for comparing data. This offers comprehensive reference data as well as other information for analysing fishes and fishery products. Independent of this, some member states are compiling their own internet data bases with the aim of improving the control of fishery products with the help of protein and DNA analysis. In Germany, for example, a catalogue with all the available data for molecular biological differentiation of fishes and fishery products has been compiled for the Federal Office for Foods and Nutrition (Bundesamt für Lebensmittel und Erhährung BLE) (http://www.fischdb.de). This data base, which is freely accessible, contains data of nearly all the standard commercial fish, mollusc and crustacean species for the following analysis methods:

It is possible to assess the nutritional status of tiny fish larvae via the volume ratio between the nucleic acids DNA and RNA.


• IEF – Iso-Electric Focusing
• SSCP - Single Strand Conformational Polymorphism
• RFLP - Restriction Fragment Length Polymorphism
• DNA - DNA-sequence of a defined area for species identification
• Alignment – Comparison of several DNA sequences

With the discovery of small and larger frauds the possibilities offered by DNA analysis are far from being exhausted, however. The methods can be put to good use in basic research and many other biological investigations, for example to clarify the genetic relationship or evolutionary biology of species or to enable systematic classification of certain species. In the case of some cyprinid species, for example, natural crosses sometimes occur if fishes spawn at the same time in the same waters. Using traditional methods it is difficult to prove whether a fish belongs to a “pure” species or whether it is a “hybrid species”. Unambiguous classification is mostly only possible on the basis of DNA analyses.

Even the nutritional condition of tiny fish larvae can be examined with the help of DNA. Here the volume ratio of the nucleic acids DNA and RNA in the body cells is used. To be able to grow, copies of the universal blueprint which is encoded in the DNA first have to be generated in the cells. This copy that in the cell serves as the matrix for the synthesis of new proteins is the RNA. The RNA molecules are not, however, only copied once but in a large number. Exactly how many copies are produced depends on the nutritional status and the metabolism of the animal. And this is reflected in the RNA/DNA ratio. In the case of starved fish RNA/DNA values are low. The more the fishes eat, the higher the RNA content becomes. Using fluorescence photometric techniques it is possible to represent the volume ratio of the two nucleic acids and thereby assess the nutritional status of the larvae.