Forensic validation of a panel of 12 SNPs for identification of Mongolian wolf and dog.

Wolf (Canis lupus) is a species included in appendices of CITES and is often encountered in cases of alleged poaching and trafficking of their products. When such crimes are suspected, those involved may attempt to evade legal action by claiming that the animals involved are domestic dogs (C. l. familiaris). To respond effectively to such claims, law enforcement agencies require reliable and robust methods to distinguish wolves from dogs. Reported molecular genetic methods are either unreliable (mitogenome sequence based), or operationally cumbersome and require much DNA (un-multiplexed microsatellites), or financially expensive (genome wide SNP genotyping). We report on the validation of a panel of 12 ancestral informative single nucleotide polymorphism (SNP) markers for discriminating wolves from dogs. A SNaPshot multiplex genotyping system was developed for the panel, and 97 Mongolian wolves (C. l. chanco) and 108 domestic dogs were used for validation. Results showed this panel had high genotyping success (0.991), reproducibility (1.00) and origin assignment accuracy (0.97 ± 0.05 for dogs and 1.00 ± 0.03 for wolves). Species-specificity testing suggested strong tolerance to DNA contamination across species, except for Canidae. The minimum DNA required for reliable genotyping was 6.25 pg/µl. The method and established gene frequency database are available to support identification of wolves and dogs by law enforcement agencies.

Distinguishing extant elephants ivory from mammoth ivory using a short sequence of cytochrome b gene.

Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated. This distinction creates opportunity for laundering and trading elephant ivory as mammoth ivory. The existing morphological and molecular genetics methods do not reliably distinguish the source of ivory items that lack clear identification characteristics or for which the quality of extracted DNA cannot support amplification of large gene fragments. We present a PCR-sequencing method based on 116 bp target sequence of the cytochrome b gene to specifically amplify elephantid DNA while simultaneously excluding non-elephantid species and ivory substitutes, and while avoiding contamination by human DNA. The partial Cytochrome b gene sequence enabled accurate association of ivory samples with their species of origin for all three extant elephants and from mammoth. The detection limit of the PCR system was as low as 10 copy numbers of target DNA. The amplification and sequencing success reached 96.7% for woolly mammoth ivory and 100% for African savanna elephant and African forest elephant ivory. This is the first validated method for distinguishing elephant from mammoth ivory and it provides forensic support for investigation of ivory laundering cases.

Influence of dietary feathers on the fecal microbiota in captive Arctic fox: do dietary hair or feathers play a role in the evolution of carnivorous mammals?

Hair and feathers are composed of keratin and are indigestible, inalimental and unpalatable for carnivores. However, carnivores often ingest hair and feathers during feeding or when grooming. We hypothesized that ingestion of hair and feathers changes species diversity and relative abundance of bacteria in the gut of carnivores. To test this hypothesis, we added disinfected poultry down feathers to the normal diet of captive Arctic foxes (Alopex lagopus). We then used fluorescently labeled terminal restriction fragments (T-RFs) to examine changes in fecal bacterial diversity and abundance. The results showed that the number of bacterial species increased significantly after feather ingestion, but that total abundance was unchanged. This demonstrated that addition of disinfected feathers to the diet stimulated increased production among less abundant bacteria, resulting in a balancing of relative abundance of different bacterial species, or that some newly-ingested microbial species would colonize the gut because a suitable microhabitat had become available. This implies that the overall production of bacterial metabolites would be made up of a greater range of substances after feather ingestion. On one hand, the host's immune response would be more diverse, increasing the capacity of the immune system to regulate gut microflora. On the other hand, the animal's physiological performance would also be affected. For wild animals, such altered physiological traits would be subjected to natural selection, and, hence, persistent geographic differences in the character of ingested feathers or fur would drive speciation.

Use of femur bone density to segregate wild from farmed Dybowski's frog (Rana dybowskii).

Wildlife has been utilized by humans throughout history and demand continues to grow today. Farming of wildlife can supplement the supply of wild-harvested wildlife products and, in theory, can reduce pressure on free-ranging populations. However, poached wildlife products frequently enter legal markets where they are fraudulently sold as farmed wildlife products. To effectively close this illegal trade in wild-captured wildlife, there is a need to discriminate wild products from farmed products. Because of the strong market demand for wild-captured frog meat and the resulting strong downward pressure on wild populations, we undertook research to develop a method to discriminate wild from farmed Dybowski's frog (Rana dybowskii) based on femur bone density. We measured femur bone density (D(f)) as the ratio of bone mass to bone volume. D(f) of wild frogs revealed a slightly increasing linear trend with increasing age (R(2)=0.214 in males and R(2)=0.111 in females, p=0.000). Wild males and wild females of age classes from 2 to ≥ 5 years had similar D(f) values. In contrast, 2-year-old farmed frogs showed significantly higher D(f) values (p=0.000) among males (mean D(f)=0.623 ± 0.011 g/ml, n=32) than females (mean D(f)=0.558 ± 0.011 g/ml, n=27). For both sexes, D(f) of wild frogs was significantly higher than that of farmed frogs (p=0.000). Among males, 87.5% (28 of 32 individuals) of farmed frogs were correctly identified as farmed frogs and 86.3% (69 of 80 individuals) of wild frogs were correctly identified as wild frogs. These results suggest that femur bone density is one reliable tool for discriminating between wild and farmed Dybowski's frog. This study also highlights a novel strategy with explicit forensic potential to discriminate wild from captive bred wildlife species.