Shane Tobe

This book provides a comprehensive and complete synopsis of forensic serology. It includes background information on different biological substances that we can detect, how the serological tests work, what the testing looks like, and how to interpret the results. Coverage includes all aspects of serological testing including basic presumptive testing, confirmatory testing, and new methods of testing, such as mRNA, methylation, proteomics, and much more.

Forensic Serology is written at a level which anyone with basic knowledge of science can fully understand and may be of particular use to undergraduate and graduate students, as well as working professionals.

Ticks are ectoparasites of major medical, veterinary and ecological importance, transmitting a wide range of pathogens to humans, companion animals and livestock. Understanding the population structure of ticks is essential for uncovering patterns of pathogen transmission, and population genetics provides a powerful method for this purpose. Tick population studies are uniquely challenging as their biology is shaped by complex interactions between hosts, microbiome and environmental factors. The choice of population genetic tools is crucial, as different methods offer varying levels of cost, throughput, resolution and accessibility, which can significantly influence the quality and scope of results. This review traces the evolution of molecular tools in tick population genetics, from early allozyme electrophoresis in the 1970s to advanced whole genome sequencing (WGS) technologies. It critically evaluates key methodologies, including allozyme electrophoresis, random amplified polymorphic DNA (RAPD), microsatellites (STRs), amplified fragment length polymorphisms (AFLP), sequence typing, restriction-site associated DNA sequencing (RADseq) and WGS, highlighting their strengths, limitations and applications. By offering a practical guide to these tools, this review helps researchers select the most appropriate methods for their studies and allows interpretation of results from older tools in the context of modern research. Sequence typing and RADseq currently provide the best balance of cost and practicality, while WGS has great potential once sequencing costs decline. This resource empowers researchers to make informed decisions, maximise the impact of their work and gain deeper insights into disease vector population structure.

Bone taphonomy and diagenesis contribute to anthropological analysis in forensic investigations by attempting to reconstruct the relationship between human cadaveric remains and their postmortem depositional environment. The rare aquatic taphonomic experiments have been delivering conflicting results on the influence of time and the environment on the decay of bone and teeth, especially considering that the main diagenetic processes can lead to fragmentation, progressive dissolution or fossilization. The aim of this experimental, quantitative, randomized and controlled 2-year study was to analyse the taphonomy and diagenesis of submerged terrestrial mammalian bones to achieve a more accurate estimation of both the post-mortem interval (PMI) and the post-mortem submersion interval (PMSI) in the short term. Three parameters of bone diagenesis, the Oxford Histological Index (OHI), the total porosity and the collagen content of cortical bone were analysed by MicroCT Scan, bright-field Light Microscopy (Picrosirius Red stain), Scanning Electron Microscopy (SEM) and Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) on 75 sheep femurs and tibias placed in four distinct types of environment (natural saltwater, natural freshwater, an artificial seawater solution and exposed to the air) vs. non-exposed controls. LA-ICP-MS was soon discontinued because no measurable changes of the elemental profiles could be detected. Multivariate statistical analysis was applied to the collected data. The macroscopical preservation was consistently excellent (OHI=5). The total porosity and the degradation of collagen were greater underwater than in subaerial exposure, whereas demineralization zones and bioerosion tunnelling appeared after 12 months in the air-exposed samples only. Underwater, the continuous movement, the correlated abrasion by sand and sediment and the constant alkaline pH (≥ 8) can explain the progressive removal of the mineral component and the subsequent exposure of collagen to bioeroders and chemical hydrolysis. On land, the same process occurs at a slower rate on account of the seasonality of the water flow, however, the action of the more abundant and diversified species of bioeroding microorganisms appears more efficient. Despite some limitations, this study indicates that three parameters of bone diagenesis can predict the depositional environment of terrestrial mammalian bone characterized by a PMI and/or PMSI of at least 12 months.

Bone and teeth, specialised bio-mineralized connective tissues, are left after the typical decomposition process of any vertebrate organism. Their analysis can reveal insights into an organism's life and retrace the history of the remains after death (also known as taphonomy), which ultimately evolves to destruction or fossilization. Studies on the taphonomy of terrestrial mammalian bio-mineralized tissues have mostly focussed on terrestrial depositional environments. Here, samples submerged in the marine environment are investigated.

Five archaeological bones of terrestrial mammalian species (pig and oxen) with historically known post-mortem submersion interval (PMSI) (69–316 years) and recovery sites, were analysed macroscopically, microscopically and by microCT. The aim was to characterize for the first time the alterations produced by marine bioeroding sponges, and to discuss their potential interdisciplinary application, with special focus on forensic investigations.

The pig samples showed microanatomical preservation (Oxford Histological Index = 3–5), increased total porosity, the presence of old tissue flakes with sponge spicules and traces of bioerosion, such as papillary holes, canals and chambers with microsculptured walls. The presence of such tissue flakes suggested that, at the time of recovery, they may have been free of sediment and inhabited by live sponges. The shape of one internal chamber was identified as the ichnospecies Entobia convoluta as typically produced by shallow, warm-water Cliothosa spp. Surface analyses for further biological evidence remained inconclusive.

The taphonomy of skeletal remains has always been relevant in anthropological, natural and forensic studies. In forensics, the role of taphonomy is to contribute to personal identification, cause of death and post-mortem interval (PMI). This study detected the past colonization of terrestrial mammalian bone by marine bioeroding sponges, and aimed to link the taphonomic findings to natural processes and environments. Bioeroding sponges are for the first time confirmed to colonize terrestrial mammalian bone submerged in marine environments, and to promote diagenesis through bioerosion.

Unlike the chemical composition and diagenetic modification of buried bones, subaqueous archaeological bone diagenesis has not been studied in detail. This observational work presents a macroscopic and microscopic characterization of 11 variably preserved archaeological terrestrial mammalian bones submerged in seawater and/or surrounded by marine sediment for 169–347 years. In situ trace element analysis was undertaken to identify geochemical fingerprints of diagenesis. The analyzed bones belong to a collection of underwater archaeological faunal materials excavated from four shipwreck sites. With one exception, all archaeological bones were fragmented, some were also heavily stained, and in two samples, the damage to the cortical layer was extensive. Bioerosion was assessed by scanning electron microscopy (SEM), and bone trace element chemistry (by laser ablation inductively coupled mass spectrometry—LA-ICP-MS) was compared with that of an unsubmerged modern sheep bone control. In the control, several trace elements were low in concentration (weighted mean concentration <1 ppm; Cr, Co, Ni, Cu, Y, rare earth elements, Th, U). In the submerged archaeological bones, the weighted mean concentration of Li, Cr, Cu, and U was enriched relative to the modern sheep bone, whereas Rb and Ba were depleted. The best-preserved bone, recovered from Batavia, showed less variation in trace element patterns compared with the more poorly preserved bones. The only archaeological bone with preserved macroscopic structure and cortex showed a gradual decrease in trace element concentration from the outer surface towards the medullary cavity, whereas in samples where more cortical damage was noted, the distribution of these elements is more irregular. With the exception of Cu and Cr, the elements focused on in this work (Li, U, Rb, and Ba) are nonessential to life, supported by their low concentration in the modern sheep bone (with the exception of Ba). The results suggest that early macroscopic and microscopic diagenetic alteration influences the concentration and distribution of chemical elements in submerged bones and that in situ trace element analysis provides clues for the reconstruction of taphonomic trajectories.

Foraminifera are marine single-celled organisms, ubiquitous in marine environments, present in brackish waters and absent in terrestrial locations. Their presence has been associated with archaeological and forensic studies only rarely, and just once and superficially with bones of terrestrial mammals. In this study, a new association is presented between foraminifera enclosed in the dissolving trabecular spaces of terrestrial mammalian bones, recovered in underwater archaeological excavations between 1968 and 1980. Research on the new association aims to detail the micro-characterization of bone in underwater environments, leading to a better understanding of bone taphonomic trajectories, the chronological sequences of changes occurring between death and the incorporation of the remains of an organism within the depositional environment. The analysis of taphonomic trajectories is known to hold relevance in distinct disciplines, such as archaeology, palaeontology and forensic sciences. Different foraminiferal taxa are linked to different marine environments, characterized by specific ranges of water depth, amount of light and oxygen, temperature and composition of sediment. The association between foraminifera and terrestrial mammalian bones indicates deposition in a marine or brackish environment, thus the analysis of the specific ecology of the identified foraminiferal taxa can point to a specific environment, adding information to paleontological, archaeological or forensics casework.

Learning Overview: The goal of this presentation is to describe the principles of bone diagenesis and their potential application to forensic science, the distinction between biogenic and diagenetic chemical signals in bones, the correlation between macroscopic and chemical characteristics of bone diagenesis, and an example of trace element analysis in a set of archaeological bones submerged for a known length of time in a marine environment. Impact on the Forensic Science Community: This presentation will impact the forensic science community by describing how the establishment of a correlation between macroscopic alterations and trace element concentrations and distributions in bones recovered from a marine environment will prove essential for members of the forensic science community involved in taphonomic assessments. Bone diagenesis is the global effect of the physical, biological, and chemical transformations that bones undergo between death and discovery in the archaeological or geological record. Diagenetic transformations, macroscopic and microscopic, are influenced by the physics, chemistry, and biology of the depositional environment. In living organisms, chemical processes are affected by diet, mobility, and pathologies. While studies of the diagenetic modifications and chemical composition of buried bones are extensively featured in the scientific literature, geochemical signatures characteristic of underwater bone diagenesis have not been studied in detail. This study investigated whether a geochemical fingerprint of the interaction between 11 archaeological mammalian bones and seawater and/or marine sediment could be discerned. The analyzed mammalian bones belong to a museum collection of underwater archaeological materials excavated from four submerged shipwreck sites off the Western Australia coast: Batavia (1629), Vergulde Draeck (1656), Zeewijk (1727) and Rapid (1811). The underwater excavations were conducted between 1968 and 1980, and bones from the four wrecks had been submerged in seawater and/or sediment for 347, 316, 241, and 169 years, respectively. With one exception, all of archaeological bones were fragmented, some were also heavily stained, and in two samples, the damage to the protective cortical layer was particularly extensive. Bone trace element chemistry was compared to that of a modern sheep bone (Ovis aries). Laser ablation-inductively coupled plasma/mass spectrometry was undertaken across bones mounted in epoxy rounds. Cross-sectional spot transverses followed a path from the cortical layer (exterior) through the trabecular bone in the interior. In the modern sheep bone, several trace elements showed bulk concentrations close to, or at, the limit of detection (Chromium [Cr], Cobalt [Co], Nickel [Ni], Copper [Cu], Yttrium[Y], Rare-Earth Element [REE], Thorium [Th], and Uranium [U]). In contrast, in the submerged bones, Lithium (Li), Cr, Cu, and U were elevated relative to the modern sheep bone, whereas Rubidium (Rb) and Barium (Ba) were depleted. Normalized trace element patterns in modern bone were flat, whereas in the archaeological samples, the normalized trace element pattern in the only whole sample (from Batavia) was different from that of the damaged bones from the other wrecks. Most elements with altered bulk concentrations in the archaeological bones are non-essential to biological life (Cu being the exception), supported by their low concentration in the modern sheep bone. However, Ba is usually enriched in bone by reason of known para-physiological metabolic processes. Since Li, Cr, Cu, U, Rb, and Ba are present in seawater in very low concentrations (<1ppm), it is reasonable to assume that in the archaeological bones, the relevant increase in bulk concentrations of Li, Cr, Cu, and U is entirely diagenetic in origin, perhaps due to protracted chemical exchange with sediment. The depletion of bulk concentrations of Rb and Ba is also diagenetic in origin and can be explained by protracted exposure to seawater and sediment. Furthermore, since the structure of cortical bone is denser than that of trabecular bone, cortical bone is less susceptible to alteration. This is reflected in the flat normalized element distribution profiles in bones where the cortical layer is missing or heavily damaged. As a consequence, the bulk chemical composition resulting from diagenetic chemical exchange in bone appears to be more uniformly distributed if the cortical layer is heavily damaged or missing, as reflected by the flat normalized elemental distribution profiles. In the only undamaged sample, the profiles of Li, Ba, Magnesium (Mg), Strontium (Sr), and Rb showed a gradual decrease in concentration from the outer surface toward the interior of the cortical bone. The overall conclusion is that macroscopic diagenetic alterations influence elemental concentrations and patterns of elemental distribution in bones, and their analysis allows the reconstruction of different taphonomic pathways.

Diagenesis is the collective word for the physical, biological, and chemical processes that bones undergo in the post-mortem period, until their physical destruction or fossilization. In forensic anthropology, the analysis of macroscopic and microscopic bone alterations, alongside the taphonomy of the soft tissues of a body, has proven valuable for the estimation of the time-of-death, or Post-Mortem Interval (PMI), of skeletonized individuals. To date, bone alterations have been mostly researched in terrestrial settings, such as exposed or buried skeletal remains, but here the scientific literature regarding human bones submerged underwater has been reviewed. It features 20 publications in the last 42 years, of which 9 are reviews, 8 are studies on ancient material and 3 are experimental studies. Future research on analysis of microscopic diagenetic parameters of submerged bones, together with the refinement of the correlation with time of the slightly better known macroscopic underwater alterations, will prove valuable for the estimation of a Post-Mortem Submersion Interval (PMSI) in both forensic and archaeological contexts, because bones have always been and still are regularly recovered underwater. The concurrent estimation of both PMI and PMSI of bones recovered underwater will add vital information to criminal investigations. Diagenetic parameters have been identified in Histological Index, protein content, porosity and crystallinity of bioapatite. They are depicted with the analytic techniques currently available to assess their presence and magnitude, and to relate them to the diagenetic processes of bioerosion, abrasion, and encrustation, but also to the extremes of dissolution or fossilization.

Wildlife crimes and the threats they present to elephant populations raise the need to develop and implement DNA-based methodology as an aid for wildlife forensic investigations and conservation efforts. This study describes the development of a tetra-nucleotide repeat STR multiplex, genotyping assay that will identify Asian elephant (Elephas maximus) and African elephant (Loxodonta africana) DNA. The assay targets six tetra-nucleotide STRs and two sex-typing markers simultaneously in both genera of elephants, a first for elephant genotyping assays. The developed assay has potential application in wildlife investigations to associate a biological sample to a particular individual elephant and additionally in conservation science for population management.

Illegal logging is one of the largest illicit trades in the world, with high profits and generally low risks of detection and prosecution. Timber identification presents problems for law enforcement as traditionally used forensic methods such as wood anatomy and dendrochronology are often unable to confidently match wood evidence to the remains of illegally felled trees. Here we have developed and validated a set of genetic markers for individualisation in bigleaf maple (Acer macrophyllum), a high value timber species often felled illegally in the USA. Using 128 single nucleotide polymorphisms and three insertion/deletion markers developed through massively parallel sequencing, 394 individuals were genotyped on the MassARRAY® iPLEX™ platform (Agena Bio-science™, San Diego, USA) to produce a population reference database for the species. We demonstrate that the resulting DNA assay is reliable, species specific, effective at low DNA concentrations (<1 ng/μL) and suitable for application to timber samples. The PID for the most common profile, calculated using an overall dataset level FST-correction factor, was 1.785 × 10−25 and PID-SIB across all individuals (treated as a single population) was 2.496 × 10-22. The further development of forensic identification assays for timber species has the potential to deliver robust tools for improved detection and prosecution of illegal logging crimes as well as for the verification of legality in reputable supply chains.