Until recently, it seemed wildlife poachers and illegal fishers could ply their trade with little or no risk of arrest, let alone being successfully prosecuted. Prior to the 1980s, Fish and Wildlife enforcement agencies charged with protecting our wildlife resources, such as the U.S. Fish and Wildlife Service, were forced to rely on scientists in universities and museums to examine evidence, rather than professional forensic scientists. Unfortunately, many of these experts had responsibilities to their institutions and were reluctant to testify in court rendering their analysis of little use (Neme 2009). Furthermore, museums and universities at the time were not equipped with adequate security measures, nor were the technicians examining the evidence maintaining the proper chain of custody required by human forensic labs.
The illegal wildlife and fisheries trade was an extremely lucrative, and up until the 1980's, low risk trade, taking several forms. According to the International Criminal Police Organization (INTERPOL) illegal trafficking of fish and wildlife is estimated to be worth in excess of $20 billion annually (Interpol 2009). According to The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) some 5,000 species of animal and 28,000 species of plant are listed as protected against international trade. Enforcement is a difficult undertaking. For example, even with local and international enforcement actions, over 4,000 African Elephants were illegally killed in 2004 alone to supply the international ivory trade (Interpol 2009). There is some legal trade in bone and ivory for other species of animals; however, it is nearly impossible to determine from what species some ivory originates by physical examination. Fortunately, a DNA technique has been developed that will determine whether or not an ivory specimen is from elephants. A recent (2006) DNA analysis of a large shipment of ivory determined that it was of elephant origin, and allowed for the seizure of 6.5 tons of illegal ivory in Singapore and another 3.9 tons in Hong Kong (Neme 2009).
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The illegal wildlife and fisheries trade is not restricted to terrestrial species. Some sturgeon eggs (a prehistoric bony fish of the family Acipenseridae) are considered a delicacy in some countries, fetching $2,000 for a 250 gram can (Neme 2009). While some of this trade is legal and the eggs are removed from sustainable populations of sturgeons, at these prices there is incentive to harvest roe from protected fish and populations. This provides an example of when forensic techniques are necessary to determine whether the trade is legal or the exploitation of a protected species.
Traditionally, much of the enforcement of wildlife protection and fisheries regulations falls to the U.S. Fish and Wildlife Service (USFWS). The National Oceanographic and Atmospheric Administration (NOAA) specifically enforces ocean fisheries regulations and performs fisheries related research. It wasn't until 1985 and again in 1989 did Congress provide funds ($1 million and $3.5 million respectively) to build an actual laboratory. This laboratory, run by the USFWS, is located in Ashland, Oregon (USFWS 2009).
Applications of DNA Technology to Fisheries and Wildlife Law Enforcement
The advent of DNA and other forensic technologies has been a boom for fisheries and wildlife scientists and law enforcement agencies. This technology allows for the testing of questioned animal parts which often are all that remains of the animal in question. For example, once fish has been skinned, cleaned, filleted, cooked, frozen, dried, or salted can be difficult to determine what species it actually is. The identity of the original organism is important as there have been many cases of one species being passed off to wholesalers and consumers as another, more valuable product. The USFWS Laboratory in Ashland, OR will analyize these samples when it is suspected that criminal fraud has occurred or conservation laws have been broken. This has also spawned a growth in the private laboratory industry. Companies such as Therion International provide analysis of fish and wildlife samples for forensic sample matching, species/subspecies differentiation, stock identification, parentage verification, individual identification, estimation of genetic variation within populations and estimation of genetic distance among populations (Therion 2009).
The application of forensic DNA typing of wildlife and fisheries samples is a relatively new endeavor. Because law enforcement must deal with a myriad of unique species, research continues to work on finding methods that can apply to both commercially important and endangered species. For example, sturgeon fish of the genus Scaphirhynchus are freshwater fish that reside in both the Mississippi and Mobile river watersheds. Two of this geneses species, S. albus, and S. suttkusi are listed as endangered, while S. platorhynchus is of commercial interest (Bemis et al 1997). The USFWS Forensic laboratory developed a method that uses a 270 base pair (bp) portion of the mitochondrial cytochrome b oxidase gene to identify sturgeon products in commercial trade (Straughan et al. 2002). Unfortunately, this method fails to distinguish S. platorhynchus from the other two members of this genus. A study by Straugham et al. illustrates the ongoing efforts to develop methods that would accurately determine speciation and assist regulators in their efforts to protect critically endangered species. In the case of Scaphirhynchus species, work is ongoing and is designed to identify useful markers. The focus currently is on 39 different loci from both mitochondrial and nuclear origins. At present, only four nuclear and one mitochondrial locus have been determined to be polymorphic (Straughan et al. 2002). Polymorphism is defined as the existence of multiple alleles of a marker at a single locus, necessary for interspecies distinction (Rudin and Inman 2002). In this case, both the nuclear and the mitochondrial markers must be used in conjunction in order be useful in forensic identification of these fish. From a logistics viewpoint, the task facing wildlife scientists and regulators is overwhelming. Given the large number of species involved, the rate at which organisms and their body parts can be transported across boarders today, plus limited resources at governmental agencies and one can easily see why the trade in poached animals and plants along with their products is such a lucrative enterprise. Unfortunately, as long as there is a demand for these products and the risk of capture low, the illegal trade will continue.
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Another way these tools are utilized is in the forensic analysis of Asian medicinal products. Traditional Asian medicine uses a wide variety of plants and herbs along with animal parts, many of which are obtained for endangered and threatened species. These would include dried seahorses, rhinoceros horn, tropical timber, and parts from Musk deer (CITES 2009). An important component in many Asian medicines are gallbladders from the bear family, Ursidae (Espinoza et al. 1994). Some North American species, specifically the American Black bear (Ursus americanus) may be hunted and their gallbladders harvested. Sport hunting of Black Bears is permitted in 27 states and all eleven Canadian provinces and territories (Neme 2009). There are numbers and season restrictions on the sport hunting of these animals. Poachers operating in the gallbladder trade operate without regard to these rules. Commercial trade in Asian bears is forbidden by most Asian countries according to CITES accords (CITES 2009).
As a result of these restrictions, much of the trade in gallbladders has shifted to North America. The gallbladders are of interest because they contain an anti-inflammatory compound Ursodeoxycholic acid, which is is now available in a synthetic form, which is not a desirable for Asian markets. Asian bear populations have suffered because of their proximity to the gallbladder trade. All have been listed by CITES in their Appendix I, which prohibits commercial trade of the live or dead animals and their parts or derivatives (CITES 2009). Because of the restriction on Asian bear part trade, gallbladders for American Black Bears have become extremely valuable, worth between $1,000 and $5,000 each (Highley 1996). Both the use of DNA fingerprinting and the use of High Performance Liquid Chromatography (HPLC), which looks specifically at the Ursodeoxycholic acid content of the bile, have allowed Fish and Game regulators and enforcement personnel to distinguish between gallbladders illegally harvested from CITES listed species and those that can be legally possessed. Both methods were developed at the USFWS Forensic Laboratory (NEME 2009).The USFWS Forensic lab now uses a multi-locus bear profiling methods specific for gallbladders. The DNA profiles of a variety of large North American mammals, birds, and fish were generated using different oligonucleotide probes (Ruth and Fain 1993). A probe is a piece of single-stranded oligonucleotide that is used in experiments to detect the prescience of a complimentary sequence of DNA found in a mixture of other single stranded DNA molecules (Rudin and Inman 2002). In the case of Black Bears, probes found to be particularly useful were 33.6, 33.15, MS1, and CMM101 (Dratch and Fain 1993). The problem with this approach however is that samples must be collected in the field and sent to Ashland, OR for analysis. Scientists at the USFWS are currently attempting to develop a field test kit what would allow field enforcement agents to quickly determine the origin of gallbladders while in the field, relying on the main laboratory for verification.
Basic Analytical Practices in Fisheries and Wildlife Forensics
As with humam crime labs, standard operating procedures (SOP) need to be employed in order to assure accuracy and precision in results, and to assure that evidence is treated in a fashion compliant with legal restrictions (Budowle et al. 2004). These SOP's are in essence standardized recipes which describe in detail the process of evidence acquisition, handling and analysis. They also specify how to interpret analytical results and preparation of the required report. As in human forensic laboratories, the biochemical assays used in the wildlife/fisheries laboratory are PCR-based (Polymerase Chain Reactor). Knowledge and experience in the use of PCR technology is important in order to forestall issues such as sample contamination. The accepted practice is to separate pre- and post-PCR operations (Butler 2005). Items such as reagents, pipettes, contamination hoods, and even protective laboratory clothing should be dedicated to pre- and post PCR areas. Again as with any laboratory it is essential to monitor Quality Control (QC) measures. It is a useful analytical technique to employ a negative control in the form of a reagent blank (Budowle et al. 2004). This is a process by which reagents used in the PCR process are run through the PCR procedure; however, the reagents are prepared without any DNA template being present. In addition to the negative control, a positive control is used, one where a sample of known type can be run with each sample or group of samples (Butler 2005).
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It is also imperative that all protocols used by the laboratory be validated to determine their reliability, accuracy, and efficacy prior to use on actual unknown samples. It is important to have a clear understanding of the operational limits of the technique (Budowle (2005). Tissues from wildlife and fisheries sources will differ in their composition and tissue matrix as compared to human tissues. While a technique may be validated and laboratory personnel have proven their proficiency in handling human samples, samples from a wildlife and fishery origin require special handling and in many cases the extraction methods for obtaining the DNA are more difficult (Neme 2009). Furthermore, one DNA has been extracted; it needs to be compared to a known. The USFWS Forensic Laboratory maintains a large database of species DNA and other data. Currently, the USFWS lab' Genetic Standards Collection contains over 40,000 samples, comprised from 800 mammal, 600 bird, 50 fish, and close to 100 amphibian and reptile species (Neme 2009). While the database is relatively large, it does not contain standards for every known species. For example, there are well over 28,000 known species of fish worldwide (Brönmark and Hansson 2005). Reptiles alone comprise over 8,700 species (Neme 2009). If the lab running wildlife samples does not have access to a known or comprehensive reference database, it can not make identifications based on the genetic material provided. Other methods, such as traditional morphological, as well as some newer isotopic methods are being investigated in order to assist in determining at least the geographic origin of evidence (Budowle 2005).
Other DNA Forensic Methods used with Wildlife and Fisheries
Wildlife and Fisheries forensic testing utilizes not only nuclear DNA samples, but as in human testing, mitochondrial DNA (mtDNA) as well. Mitochondrial DNA is passed to offspring only through the maternal line, and has been used in animals for species identification, population assignment and geography, verification of food products and alteration, identification of animal fibers in clothing, and of course, the identification of poaching (Budowle 2005). Again, researchers tend to focus on the mitochondrial cytochrome b oxidase gene. This technique is particularly useful for hair analysis, and is used when fibers are found at crime scenes (especially when determining the type of animal present at a crime scene) or when animal products are suspected to contain hairs from species listed as endangered or threatened. It is also useful when looking at geographic differences in organisms.
Some populations of fish or wildlife may not be considered threatened while others in specific region may be on the verge of extinction. This is an excellent technique in determining evidence in genetic isolation and reproductive segregation. MtDNA analysis is useful in the determination of organisms' geographic origins, and has been especially useful in the enforcement of fish and tropical timber regulations (Brunham et al. 1999).
Many fisheries and wildlife populations are facing extinction due to habitat loss, reproductive and genetic isolation, the effects of climate change, and the illegal poaching of their populations (Falk et al. 2006). Fortunately the advances in forensic technologies has permitted law enforcement to mount a counter attack based on sound science, which in tern assists prosecutors in winning cases and providing a real deterrent to these activities. Both figure 1 and 2 below were taken at the California Fish and Game Department Frozen Storage facility at Comanche Lake in San Joaquin County, California. Both pictures depict animals that were harvested illegally and are being stored for further forensic analysis and prosecution of the alleged offenders.
Not long ago, poachers would have escaped unprosecuted due to lack of reliable evidence, or if convicted, received only a minor fine and little or no jail time. Advancements in DNA testing and other forensic tools are resulting in a higher rate of conviction. As a result, species such as walrus, elephants, and sturgeon, are receiving greater protection, as are specific populations that are listed as endangered or threatened. History has shown that when the value of wildlife is judged by economics alone, it continues to be exploited, resulting in the lost of species and biodiversity (Neme 2009). Fortunately, increased use of forensic techniques can provide valuable tools to those who seek to protect valuable and diminishing living resources.
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- CITES 2009. Committee on International Trade in Endangered Species of Wild Fauna and Flora. www.CITES.org. Accessed online November 28, 2009
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- Espinoza, E.P., Mary-Jacque Mann, Bonnie Yates, and Larry Elliot. 1994. Preliminary forensic analysis of the wildlife content of patented Asian medicines. Endangered species and patented oriental medicines in trade: A Traffic Network Report. World Wildlife Fund.
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- (Interpol).International Criminal Police Organization, 2009Wildlife Crime. http://www.interpol.int/Public/EnvironmentalCrime/Wildlife/Default.asp. Accessed online November 30, 2009
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- (USFWS) United Stated Fish and Wildlife Service Forensic Laboratory. 2009.http://www.lab.fws.gov. Access online November 29, 2009
- (Therion) Therion International. 2009. DNA Testing for Fish & Wildlife Conservation, Forensics and Management. www.theriondna.com/pdf/wildlife.pdf. Accessed online December 8, 2009.