Nicole E. White

Deep Sequencing of Plant and Animal DNA Contained within Traditional Chinese Medicines Reveals Legality Issues and Health Safety Concerns

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant reference databases become better established.

DNA-Based Faecal Dietary Analysis: A Comparison of qPCR and High Throughput Sequencing Approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.

Scrapheap Challenge: A novel bulk-bone metabarcoding method to investigate ancient DNA in faunal assemblages

Highly fragmented and morphologically indistinct fossil bone is common in archaeological and paleontological deposits but unfortunately it is of little use in compiling faunal assemblages. The development of a cost-effective methodology to taxonomically identify bulk bone is therefore a key challenge. Here, an ancient DNA methodology using high-throughput sequencing is developed to survey and analyse thousands of archaeological bones from southwest Australia. Fossils were collectively ground together depending on which of fifteen stratigraphical layers they were excavated from. By generating fifteen synthetic blends of bulk bone powder, each corresponding to a chronologically distinct layer, samples could be collectively analysed in an efficient manner. A diverse range of taxa, including endemic, extirpated and hitherto unrecorded taxa, dating back to c.46,000 years BP was characterized. The method is a novel, cost-effective use for unidentifiable bone fragments and a powerful molecular tool for surveying fossils that otherwise end up on the taxonomic “scrapheap”.

Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Egg forensics: An appraisal of DNA sequencing to assist in species identification of illegally smuggled eggs

Abstract Psittaciformes (parrots and cockatoos) are charismatic birds, their plumage and capacity for learning make them highly sought after pets. The illegal trade in parrots and cockatoos poses a serious threat to the viability of native populations; in addition, species transported to non-endemic areas may potentially vector disease and genetically ‘pollute’ local native avifauna. To reduce the logistical difficulties associated with trafficking live birds, smugglers often transport eggs. This creates a problem for authorities in elucidating accurate species identification without the laborious task of incubation and hand rearing until a morphological identification can be made. Here, we use 99 avian eggs seized from carriers coming into and within Australia, as a result of suspected illegal trade. We investigate and evaluate the use of mitochondrial DNA (mtDNA) to accurately identify eggs to family, genus or species level. However, Identification of a species based on percentage mtDNA similarities is difficult without good representations of the inter- and intra-levels of species variation. Based on the available reference database, we were able to identify 52% of the eggs to species level. Of those, 10 species from eight genera were detected, all of which belong to the parrot (Psittacidae) and cockatoo (Cacatuidae) families. Of the remaining 48%, a further 36% of eggs were identified to genus level, and 12% identified to family level using our assignment criteria. Clearly the lack of validated DNA reference sequences is hindering our ability to accurately assign a species identity, and accordingly, we advocate that more attention needs to be paid to establishing validated, multi locus mtDNA reference databases for exotic birds that can both assist in genetic identifications and withstand legal scrutiny.

Application of STR markers in wildlife forensic casework involving Australian black-cockatoos (Calyptorhynchus spp.)

Parrots and cockatoos are highly prized aviary birds and the demands for such species has fuelled their illegal trade and harvest from the wild. Here we report on three forensic case studies involving black-cockatoos (Calyptorhynchus spp.) endemic to Australia. These cases involve suspected poaching and illegal killing of endangered red- and white-tailed black-cockatoos. Through the prior development of 20 polymorphic microsatellite loci and population databases for white- and red-tailed black-cockatoos, the tools are available to conduct high-resolution paternity and individual identity testing. In one case, we matched a red-tailed black-cockatoo nestling to a tree hollow from which it was poached through the use of DNA from eggshell recovered from the nest. For the second case, we utilized our provenance population database (nest sites), and identified the kinship and geographic origin of a white-tailed black-cockatoo, which was illegally harvested from the wild. The third case determined the number individual white-tailed black-cockatoos allegedly shot at a fruit growers orchard from body part remains. These genetic investigations highlight the significance and statistical confidence of DNA profiling and associated databases for endangered taxa, such as exotic birds. Our cockatoo population databases are the first of their kind in Australia, and demonstrate the efficacy of such approaches to identify such illegal activity. With a robust set of genetic markers and methodologies in place, we aim to broaden our population databases to include other cockatoo species of conservation concern.

Metabarcoding avian diets at airports: implications for birdstrike hazard management planning.

BackgroundWildlife collisions with aircraft cost the airline industry billions of dollars per annum and represent a public safety risk. Clearly, adapting aerodrome habitats to become less attractive to hazardous wildlife will reduce the incidence of collisions. Formulating effective habitat management strategies relies on accurate species identification of high-risk species. This can be successfully achieved for all strikes either through morphology and/or DNA-based identifications. Beyond species identification, dietary analysis of birdstrike gut contents can provide valuable intelligence for airport hazard management practices in regards to what food is attracting which species to aerodromes. Here, we present birdstrike identification and dietary data from Perth Airport, Western Australia, an aerodrome that saw approximately 140,000 aircraft movements in 2012. Next-generation high throughput DNA sequencing was employed to investigate 77 carcasses from 16 bird species collected over a 12-month period. Five DNA markers, which broadly characterize vertebrates, invertebrates and plants, were used to target three animal mitochondrial genes (12S rRNA, 16S rRNA, and COI) and a plastid gene (trnL) from DNA extracted from birdstrike carcass gastrointestinal tracts.ResultsOver 151,000 DNA sequences were generated, filtered and analyzed by a fusion-tag amplicon sequencing approach. Across the 77 carcasses, the most commonly identified vertebrate was Mus musculus (house mouse). Acrididae (grasshoppers) was the most common invertebrate family identified, and Poaceae (grasses) the most commonly identified plant family. The DNA-based dietary data has the potential to provide some key insights into feeding ecologies within and around the aerodrome.ConclusionsThe data generated here, together with the methodological approach, will greatly assist in the development of hazard management plans and, in combination with existing observational studies, provide an improved way to monitor the effectiveness of mitigation strategies (for example, netting of water, grass type, insecticides and so on) at aerodromes. It is hoped that with the insights provided by dietary data, airports will be able to allocate financial resources to the areas that will achieve the best outcomes for birdstrike reduction.

Characterisation and cross-species utility of 20 microsatellite markers for population and forensic applications in the endangered Carnaby’s Black-cockatoo, Calyptorhynchus latirostris

We characterise 20 microsatellite loci identified from the endangered Carnaby’s Black-cockatoo (Calyptorhynchus latirostris). The primers were tested across 40 individuals from the southwest of Western Australia and displayed between 4 and 11 alleles per locus with expected heterozygosities ranging from 53 to 87% and exclusion probabilities of ≥0.999. These loci will be useful in population genetic studies to facilitate conservation management decisions in addition to wildlife enforcement applications for the endangered Carnaby’s Black-cockatoo. We also tested the markers in 12 high profile and smuggled species from five genera, Cacatua, Callocephalon, Calyptorhynchus, Nymphicus and Probosciger. These species detected between 2 and 19 alleles per locus with 50–100% amplification success.