Ancient DNA & stable isotope profiling of fossil eggshell

Eggshell is frequently encountered within palaeontological and archaeological deposits and due to various chemical and physical properties, it is highly valued as a substrate for geochronology, palaeodietary and palaeoecological applications. Despite the wellrecognised preservation of biomolecules in fossil eggshell, the substrate had never been evaluated for ancient DNA (aDNA) preservation. Herein, this thesis describes the first exploration involving the recovery and characterisation of aDNA from fossil eggshell. The primary aim of this thesis research is to explore eggshell aDNA and how genetic data, when combined with stable isotope profiles, provides new insights into hunting practices, zooarchaeology, palaeodiets, palaeobiology and extinction processes. The visualisation of fluorescently-stained DNA in fossil eggshell by confocal microscopy revealed that genetic material was present in the eggshell of the extinct New Zealand moa (Aves: Dinornithiformes) and Madagascan elephant bird (Aves: Aepyornithiformes). The immediate task was to determine how to go about characterising the aDNA encapsulated in this previously unrecognised substrate. Quantitative PCR assays were employed to thoroughly evaluate different methods to maximise the recovery and amplification of aDNA from powdered eggshell. This study resulted in the successful characterisation of both mitochondrial and nuclear DNA isolated from 19,000 year-old emu eggshell and the first ever DNA sequence from the elephant bird, Aepyornis. All data pointed toward fossil eggshell as a reliable source of relatively pure aDNA. This work culminated in a formal description of the methodology that should be employed to maximise recovery of aDNA from eggshell. Next, this thesis investigates whether eggshell aDNA could be applied to ‘first-contact’ archaeological sites in New Zealand, to gain new insights into the Moa-hunter period of prehistory. The abundance of moa eggshell within the ‘first-contact’ Polynesian archaeological sites is well recognised from previous excavations. With nine species of moa contained within the New Zealand archipelago however, the scientific value of these remains has been limited by the lack of definitive species assignments. Genetic species assignments of moa eggshell combined with morphological identification of bone (literature and museum catalogued specimens) has enabled the most comprehensive audit of zooarchaeology assemblages from several significant 13th-15th century AD deposits. In total, mitochondrial DNA (mtDNA) was amplified from 251 of 323 (78 %) eggshell fragments and 22 of 27 (88 %) bone samples, and the analyses revealed the presence of four moa species: Anomalopteryx didiformis; Dinornis robustus; Emeus crassus; and Euryapteryx curtus. The mtDNA, together with highly polymorphic microsatellite markers, were used in combination to individualise eggshell fragments. These data revealed over 50 individual eggs from Wairau Bar – a number that likely represents a considerable proportion of the total reproductive output of moa in the area. This emphasises that human predation of all life stages of moa was intense. In addition, an excess of male bones at the Wairau Bar site (determined by molecular sexing) suggests that males might have been preferential targets, perhaps while incubating eggs. Lastly, moa eggshell genetics and stable isotope profiles were combined to examine the palaeodiets, palaeobiology, palaeoecology and the human mediated extinction of the moa. Organic (δ13Corg and δ15N) and carbonate (δ13Ccarb and δ18O) stable isotope profiles were analysed from 247 genetically identified eggshell fragments. The combination of ancient DNA and stable isotope profiles revealed moa consumed a diet consisting exclusively of C3 vegetation and occupied the varied landscape that surrounded the archaeological deposits. Given that moa were foraging in such diverse landscapes suggests that the first colonisers hunted these moa away from the homogenous landscapes immediately surrounding these archaeological sites and thus engaged in ‘central point foraging’. Discriminant functional analyses indicate moa eggshells can be assigned with good precision to their parent species based purely on their stable isotope profiles. In conclusion, this thesis research has revealed a ‘new’ aDNA substrate that has a number of useful applications in archaeology, palaeogenomics and palaeoecology. The research on New Zealand moa eggshell demonstrated, first-hand, how this substrate can provide valuable insights into the biology, ecology and extinction of a megafaunal species. It is hoped the research will have a lasting influence on eggshell discovered across the globe

Molecular Characterization of Haemaphysalis Species and a Molecular Genetic Key for the Identification of Haemaphysalis of North America

Haemaphysalis longicornis (Acari: Ixodidae), the Asian longhorned tick, is native to East Asia, but has become established in Australia and New Zealand, and more recently in the United States. In North America, there are other native Haemaphysalis species that share similar morphological characteristics and can be difficult to identify if the specimen is damaged. The goal of this study was to develop a cost-effective and rapid molecular diagnostic assay to differentiate between exotic and native Haemaphysalis species to aid in ongoing surveillance of H. longicornis within the United States and help prevent misidentification. We demonstrated that restriction fragment length polymorphisms (RFLPs) targeting the 16S ribosomal RNA and the cytochrome c oxidase subunit I (COI) can be used to differentiate H. longicornis from the other Haemaphysalis species found in North America. Furthermore, we show that this RFLP assay can be applied to Haemaphysalis species endemic to other regions of the world for the rapid identification of damaged specimens. The work presented in this study can serve as the foundation for region specific PCR-RFLP keys for Haemaphysalis and other tick species and can be further applied to other morphometrically challenging taxa

Perception of Ticks and Tick-Borne Diseases Worldwide

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges

Molecular and morphological analyses of avian eggshell excavated from a late thirteenth century earth oven

Using ancient DNA (aDNA) extracted from eggshell of the extinct moa (Aves: Dinornithiformes) we determined the species composition and number of eggs found in a late thirteenth century earth oven feature at Wairau Bar (South Island, New Zealand) - one of New Zealand's most significant archaeological sites. Mitochondrial and nuclear DNA signatures confirmed this oven feature contained fragments of at least 31 moa eggs, representing three moa genera: Emeus; Euryapteryx; Dinornis. We demonstrate through the genetic identification of 127 moa eggshell fragments that thickness is an unreliable character for species assignment. We also present a protocol for assessing the preservation likelihood of DNA in burnt eggshell. This is useful because eggshell fragments found in archaeological contexts have often been thermally modified, and heat significantly increases DNA fragmentation. Eggshell is widely used in radiocarbon dating and stable isotope research, this study showcases how aDNA can also add to our knowledge of eggshell in both archaeological and palaeoecological contexts

Ticks and Tick-borne Diseases

Lyme borreliosis (or Lyme Disease) is an emerging threat to human health in the Northern Hemisphere caused by tick-borne bacteria from the Borrelia burgdorferi sensu lato (Bbsl) complex. Seabirds are important reservoir hosts of some members of the Bbsl complex in the Northern Hemisphere, and some evidence suggests this may be true of penguins in the Southern Hemisphere. While the Bbsl complex has not been detected in Australia, a novel Borrelia species (‘Candidatus Borrelia tachyglossi’) was recently sequenced from native ticks (Ixodes holocyclus and Bothriocroton concolor) parasitising echidnas (Tachyglossus aculeatus), suggesting unidentified borreliae may be circulating amongst native wildlife and their ticks. In the present study, we investigated whether ticks parasitising little penguins (Eudyptula novaehollandiae) harbour native or introduced Borrelia bacteria. We chose this penguin species because it is heavily exploited by ticks during the breeding season, lives in close proximity to other potential reservoir hosts (including native wildlife and migratory seabirds), and is known to be infected with other tick-borne pathogens (Babesia). We screened over 230 penguin ticks (Ixodes spp.) from colonies in south-eastern Australia, and found no evidence of Borrelia DNA. The apparent absence or rarity of the bacterium in south-eastern Australia has important implications for identifying potential tick-borne pathogens in an understudied region.This study was part funded by the Murdoch University School of Veterinary and Life Sciences small grant schem

Distribution and Prevalence of <i>Theileria orientalis</i> Genotypes in Adult Lactating Dairy Cows in South West Region of Western Australia

Bovine anaemia caused by Theileria orientalis group (BATOG) causes significant production and economic losses in Australia’s cattle industry. The pathogenic T. orientalis genotypes reported in Australian cattle are type 1 (Chitose) and type 2 (Ikeda). The present study aimed to determine the prevalence and distribution of T. orientalis genotypes in adult lactating cows in Western Australia (WA) dairy herds. A total of 100 whole blood samples from lactating cows from 10 farms were obtained and screened for T. orientalis using polymerase chain reaction (PCR). Sanger sequencing was subsequently used to characterise T. orientalis genotypes isolated from positive samples. A total of thirteen cows (13%; 95% CI: 7.1–21.2%) were positive for T. orientalis, and six out of ten farms (60%; 95% CI: 26.2–87.8%) housed at least one T. orientalis-positive cow. The distribution of T. orientalis was found to be wide and dense in the South west region of WA and the southern coast of WA. The predominant T. orientalis genotype identified was Ikeda (n = 11, 11%; 95% CI: 5.6–18.8%), while the Buffeli genotype was identified in WA for the first time, albeit at a low prevalence (n = 1, 1%; 95% CI: 0.0–5.4%). This study has provided useful epidemiological evidence on the prevalence and distribution of T. orientalis in adult lactating dairy cows in WA dairy farms, and on the importance of conducting widespread surveillance programs for the understanding of BATOG in WA

Recent insights into the tick microbiome gained through next-generation sequencing

Abstract The tick microbiome comprises communities of microorganisms, including viruses, bacteria and eukaryotes, and is being elucidated through modern molecular techniques. The advent of next-generation sequencing (NGS) technologies has enabled the genes and genomes within these microbial communities to be explored in a rapid and cost-effective manner. The advantages of using NGS to investigate microbiomes surpass the traditional non-molecular methods that are limited in their sensitivity, and conventional molecular approaches that are limited in their scalability. In recent years the number of studies using NGS to investigate the microbial diversity and composition of ticks has expanded. Here, we provide a review of NGS strategies for tick microbiome studies and discuss the recent findings from tick NGS investigations, including the bacterial diversity and composition, influential factors, and implications of the tick microbiome