Source: Addendum to Proposal submitted by Norway to transfer the northeast Atlantic and North Atlantic Central stocks of minke whale Balaenoptera acutorostrata from Appendix I to Appendix II. Letter from the Norwegian Directorate for Nature Management to the CITES Secretariat, 28 May, 1997.

ADDENDUM

to Proposal submitted by Norway to transfer the northeast Atlantic and North Atlantic Central stocks of minke whale Balaenoptera acutorostrata from Appendix I to Appendix II.


Executive summary

Norway is presently developing a control system aiming at inter alia detecting any attempts at illegal trade in products from other stocks of minke whales or other species of baleen whales. The key elements in this control system are DNA samples taken of each animal caught under the Norwegian catch quota, analyses and registering of DNA-profiles for whales in a database.

The whale DNA profile entered in the register will be composed of three parts:

The mitochondrial DNA and Y chromosome DNA primarily provide information on species and stocks for samples where the sampled animal is not in the register.

These types of DNA profiles are reproducible between various laboratories and countries. They can be digitized and entered in a searchable database. The technology needed is well-known and used in relevant countries. Thus it is possible to communicate profiles unambiguously between countries without transfer of samples.

The register could be operative from early 1998, starting with DNA profiles of the minke whales caught during the 1997 season. Thereafter profiles of all legally caught whales will be entered in the register, as well as profiles from other minke whale stocks and from other whales species to the extent that such samples will be available (e.g. from biopsy sampling).

Such a register system will be fully transparent and will be useful to all authorities involved in controlling the distribution of whale products. Meat or blubber can be sampled in the marked, and the DNA profile can be determined in a local laboratory. If the meat or blubber stem from a legally-caught Norwegian whale, it will always be possible to identify the individual from which the sample is taken. If the DNA profile is not found in the register, it will often be possible to identify the stock and /or species from which the sample is taken.

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1 Eight highly polymorphic tetranucleotide STRs.
2 mDNA: sequence of 400 base pairs from the most polymorphic part of the control region.
3 Y-DNA: four STRs.

GENETIC LABELLING OF WHALE MEAT
REPORT AND PROPOSALS FOR THE ESTABLISHMENT OF A DNA REGISTER FOR WHALES

by
Professor Bjørnar Olaisen, Institute of Forensic Medicine, University of Oslo

BACKGROUND
My relevant scientific qualifications in this context are in the field of human forensic genetics. I am in charge of forensic genetics at the Institute of Forensic Medicine, University of Oslo. The Institute is responsible for DNA diagnostics in paternity cases on a national basis, and also undertakes DNA analyses of biological evidence for the police throughout the country. (See e.g. "Identification by DNA analysis of the victims of the August 1996 Spitsbergen civil aircraft disaster", Nature Genetics 1997, 5(4): 402-405). DNA registers have been established as part of the criminal justice system in a number of countries, and Norway is also to establish one in response to recommendations by an official committee (NOU 31 (1993)). For several years, work has been in progress to draw up European guidelines for such registers, particularly through the work of the European DNA Profiling Group (EDNAP). They are intended to be used in criminal proceedings, when the DNA profile of biological material found at the scene of the crime can be compared with the profiles of convicted criminals which are recorded in the register. The purpose of such registers is thus very similar to that of a whale register: this would make it possible to compare the DNA profile of whale meat or blubber bought on the open market with the profiles in the register. If the meat or blubber is from a legally-caught Norwegian minke whale, it will be possible to identify the individual from which the sample is taken. By comparing the DNA profile of biological material from an unknown individual with the profiles in a register of known individuals, it is possible to find out whether the unknown individual is in the register, and if so, to identify him.

In this connection, biological differences between humans and whales are of little importance. We have far more detailed information on the human DNA molecule than is the case for whales, but the factors behind the choices of methods made for human DNA registers will in general also be applicable to such registers for whales.

REQUIREMENTS FOR DNA PROFILES FOR USE IN REGISTERS

1. DNA profiles for use in the register must be composed in a way that ensures they are so characteristic/individual that a match between two profiles in practice proves that they are from the same individual

2. It must be possible to digitize the DNA profiles and enter them in a searchable register.

3. The DNA profiles must be possible to reproduce so accurately that the profile in the register is practically always the same as that obtained by drawing up profiles from other samples from the same individual.

4. DNA profiles should also normally be reproducible between various laboratories and countries. This means that the technology needed should be well-known and used in other relevant countries/laboratories, and that it should be possible to communicate profiles unambiguously between laboratories and countries (nomenclature, etc.).

OTHER DESIRABLE PROPERTIES OF THE DNA PROFILE

In addition to being so specific that they can be used to identify individuals, the DNA profiles used for this purpose should be so specific to species and stock that they provide reasonably reliable information at this level as well. In cases where the profile is found in the register, this will not be of any great importance, but if it is not in the register, it will be useful to establish whether the profile belongs to a minke whale and if so from which stock.

CHOICE OF COMPONENTS IN THE DNA PROFILE

The first step is to choose the type of genetic variation at molecular DNA that best meets the requirements described above. For practical purposes, three options currently exist:


Minisatellites: Both multilocus and single locus analyses of whale tissue have been carried out, and both have been shown to give a information that is highly specific to the individual, although I know of only one good published SLP probe. Multilocus analyses (Jeffreys' probes 33.15/33.6, the alphaglobin probe, etc) are an extremely effective techniques for "side-by-side" comparison. However, no generally accepted method for digitizing or communicating multilocus profiles has been established, and they are therefore not appropriate for use in a register. SLP analyses can be digitized, but experience shows that inter-laboratory variation in the results can be large, which reduces their efficiency for the purpose of a register. One special type of analysis (MVR analysis) can give profiles that are highly specific to the individual, but the method is not (yet) widespread. In conclusion, I recommend that minisatellites should not be used for the DNA register.

Microsatellites: Microsatellite analyses have also been used for tissue samples from whales, including minke whales. As in other animal species, the level of individual variation is high. To my knowledge, only dinucleotide STR analyses (CA repeat analyses) have been carried out on whales, but there is no reason to believe that the level of variation would be any lower for tetranucleotide STRs in whales. Tetranucleotide STRs are preferred in human material for forensic genetic purposes. This is because they give more reliable typing than dinucleotide STRs (the difference between alleles is at least four base pairs and there is little tendency for "stutter bands" to appear when using tetranucleotide STRs, whereas for dinucleotide STRs the difference between alleles may be as small as two base pairs, and there is a marked tendency for "stutter bands" to appear). In forensic genetics, it has been found that reproducibility and international communicability are good for tetranucleotide STRs. The technology is also in widespread use. The use of this technique would require a preliminary effort to detect and evaluate appropriate STRs. It would probably be possible to adopt the technique described by the Utah Marker Development Group (A Collection of Ordered Tetranucleotide-Repeat Markers from the Human Genome, Am J Hum Genet 57 619-628, 1995, and references given in the article). For example, AGAT repeats could be used. and loci selected that are robust, simple (no allele differences < 4 bp) and polymorphic. If a set of eight STRs with a mean discriminating power, DP, of 96 % (corresponding to 87 % heterozygosity) could be found, the probability of two individuals being identical in all eight systems would be of the order of 10-11. The probability of any two individuals in a stock of 100 000 animals having identical profiles would thus be low. Profiles of this type could also be used to detect and establish most parent/offspring relationships between the individuals in the register - at least if mtDNA data (for mother/offspring relationships) or Y chromosome data (for father/son relationships) are also available.

Y chromosome STRs: As far as I know, no Y chromosome STRs from whales have been published. Such markers could be very valuable, together with mtDNA information, as regards information on species and stocks. However, a great deal of preliminary work might be required to find such polymorphisms in whales. Although I am not a specialist in this field, I assume that it would be possible to find such variation relatively simply by using the human DNA sequence as a basis for PCR-based screening for polymorphism in the Y chromosome in whales. A Norwegian group has published a DNA-based method for gender determination in whales, including minke whales (Palsbøll, P.J., A. Vader, I. Bakke and M.R. El-Gewely: Determination of gender in cetaceans by the polymerase chain reaction, Can J Zool 70 2166-2170 (1992)).

mtDNA: The entire mitochondrial genome of whales has been sequenced, and papers have been published demonstrating wide variation both between species and within species and stocks. One paper (Bakke, I., S. Johansen, Ø. Bakke and M. R. El-Gewely: Lack of population subdivision among the minke whales (Balaenoptera acutorostrata) from Icelandic and Norwegian waters based on mitochondrial sequences, Marine Biol. 125 1-9, 1996) shows substantial individual variation in the North Atlantic minke whale, even though one haplotype was found in 20 of 87 animals. The remaining animals were fairly evenly distributed among 24 haplotypes. As far as we know, mtDNA from North Atlantic minke whales is always clearly different from that of other whales, and so far has always proved to be different from mtDNA from Antarctic minke whales, which can be divided into two stocks of quite different types. Thus, the necessary conditions are met and the technology is available. The method is based on DNA sequence data, which can easily be digitized and communicated, and the technology is available "everywhere" (and the procedures are partly automated).

PROPOSED COMPONENTS OF THE DNA PROFILE ANALYSIS FOR THE WHALE REGISTER

1. A set of tetranucleotide STRs with the following characteristics:
About eight STRs, robust, simple and highly polymorphic (heterozygosity > 0.8). These should be possible to include in two multiplex analyses (in the PCR analysis and in the fragment analysis).
2. mtDNA sequencing of the most polymorphic part of the control region (ca 400 base pairs).
3. Two to four good Y chromosome STRs as soon as they are available (one amplification, one fragment analysis).

A DNA profile of this kind would be extremely effective. The eight STRs would characterize an individual so well that it would be most unlikely that any particular profile would occur more than once even in a relatively large stock. The mtDNA profile alone would probably provide a reliable basis for species identification and a reasonable basis for suggesting the stock from which a sample originates. The eight STRs would provide some additional information for this purpose, and if Y chromosome markers can also be included, it would probably be possible to identify the stock definitely.

PROPOSAL FOR ORGANIZATION OF THE REGISTER

In the USA and UK, DNA registers for forensic purposes are run by the large police organizations (the FBI and the Home Office's Forensic Science Services), which both carry out typing for the registers and administer them (searching, security procedures, permits, etc). It appears that Sweden intends to follow a similar pattern. In Norway, the Institute of Forensic Medicine is to be responsible for laboratory work and supplying the profiles to the register, while the National Bureau of Crime Investigation will be responsible for administration of the register. The reasons behind the choice of this system for the human DNA register in Norway also apply to a whale DNA register: the people who have the scientific expertise to carry out the laboratory work do not have the necessary legal expertise to run the register, and vice versa. In my opinion, separation of the two functions will also be the best way of showing the general public that the system is fair and impartial.

My proposal is therefore that the Ministry should appoint the best-qualified laboratory to carry out DNA typing and supply DNA profiles, preferably after inviting tenders. The register should be administered by a central public institution with the necessary legal expertise.

The Ministry of Justice, the National Bureau of Crime Investigation and Institute of Forensic Medicine can provide valuable assistance during the practical work of establishing and organizing the register. The National Bureau of Crime Investigation (and the Institute of Forensic Medicine, which is to supply the profiles) are currently making decisions as regards computer technology for the human DNA register, and these can also be used for the whale register, if appropriate. Similarly, it would be possible to coordinate quality assurance for both the profiles and the registers. However, since mtDNA is not to be used in the human register, it will be necessary to adapt the computer system for this purpose. The computer system is expected to be relatively simple, so that investment costs will be low. In the near future, practical use of the register is only expected to require very limited manpower.

PROPOSED SCHEDULE

I believe it is realistic to envisage the establishment of an operative register from 1 January 1998, which would include whales caught in 1997. To achieve this, I propose the following strategy:

Pilot projects
Two pilot projects should be started immediately, and these should be run by the laboratories which are currently best equipped for the projects and therefore do not require much time for preparation:

1. One project must be started to establish the eight STRs. This work should be done by a laboratory which can construct and screen an M13 genomic library, sequence clones and use material from 50 animals to test tetranucleotide STRs for robustness and degree of polymorphism. Towards the end of this pilot project, the choice of STRs and various methodological choices which are of particular importance as regards quality assurance (choice of multiplex, colour combinations, internal standards, etc) should be coordinated with the corresponding choices for the human register.

Assessment of costs: I estimate that to construct and screen an M13 genomic library, sequence about 30 clones of 500 base pairs, purchase primers, test PCR products and type 50 animals in about 20 STRs will require one full-time employee for six months.

Time frame: Six months.

2. Y chromosome markers will have to be established more from first principles, since there appears to be no previous work on Y chromosome STRs in minke whales. A first approach should be made on the basis of human Y-chromosome polymorphisms. If this simple approach does not reveal the desired markers, it would perhaps be wise to discontinue the project until more progress has been made internationally in research on Y chromosome markers in whales.

 

Assessment of costs: Difficult to assess, but if the approach of using human Y-chromosome research is successful, the goal might be achieved by one full-time employee in two or three months.

Implementation
Once the pilot projects have been completed, the results must be evaluated and analysed statistically. The final choice of components for the profile can then be made, together with procedures and quality assurance measures. Tenders for the supply of DNA profiles for the register can be invited from relevant laboratories and institutions, and the register can be operative from 1 January 1998, when typing of the 1997 catch can begin.

OTHER BENEFITS OF THE PROJECT AND REGISTER

I do not intend to go into much detail here, partly because whale biology in the broadest sense is outside my field of special expertise. However, there can be little doubt that data used in a DNA profile including so many animals and intended to cover a long period of time can be of great value to the research circles involved in the work, and to whale researchers in general if the data are made generally available. The degree of kinship between animals in the catch could be useful in estimating stock sizes. The profile will give us an unprecedented amount of information for such purposes.

LABORATORIES CONSIDERED TO BE POTENTIAL CANDIDATES FOR SUPPLYING DNA PROFILES TO THE REGISTER

I have contacted various institutions where there are research groups with a particular interest in fish and whale biology. All these groups were interested in being given the opportunity to take part in a project concerning a DNA register for whales caught by Norwegian vessels. During the conversations, I used a check-list of questions in order to find out how closely the interests and research profiles of the groups coincided with the recommendations I have made above.

I suggest that the issue of which group(s) should be approached with a view to taking on tasks connected with the register on a permanent basis should be considered once the pilot projects are under way or completed.

A project of limited scope has been started at the Institute of Forensic Medicine. One of its aims is to check that there are no problems associated with DNA analyses of blubber. The results show that DNA can readily be extracted from blubber.

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