Who bit the boat? New DNA collection and genomic methods enable species identification in suspected shark-related incidents.

Species identification following shark-related incidents is critical for effective incident management and for collecting data to inform shark-bite mitigation strategies. Witness statements are not always reliable, and species identification is often ambiguous or missing. Alternative methods for species identification include morphological assessments of bite marks, analysis of collected teeth at the scene of the incident, and genetic approaches. However, access to appropriate collection media and robust genetic assays have limited the use of genetic technologies. Here, we present a case study that facilitated a unique opportunity to compare the effectiveness of medical gauze readily available in first-aid kits, and forensic-grade swabs in collecting genetic material for shark-species identification. Sterile medical gauze and forensic-grade swabs were used to collect transfer DNA from the bite margins on a bitten surf ski which were compared to a piece of shark tissue embedded along the bite margin. Witness accounts and the characteristics of the bite mark impressions inferred the involvement of a Carcharodon carcharias (white shark). The morphology of a tooth found on the boat that picked up the surf ski, however, suggested it belonged to an Orectolobus spp. (wobbegong). Genetic analysis of DNA transferred from the shark to the surf ski included the application of a broad-target nested PCR assay followed by Sanger sequencing, with white shark contribution to the 'total sample DNA' determined with a species-specific qPCR assay. The results of the genetic analyses were congruent between sampling methods with respect to species identification and the level of activity inferred by the donor-specific DNA contribution. These data also supported the inferences drawn from the bite mark morphology. DNA from the recovered tooth was PCR amplified with a wobbegong-specific primer pair designed for this study to corroborate the tooth's morphological identification. Following the confirmation of gauze used for sampling in the case study event, two additional isolated incidents occurred and were sampled in situ using gauze, as typically found in a first-aid kit, by external personnel. DNA extracted from these gauze samples resulted in the identification of a white shark as the donor of the DNA collected from the bite marks in both instances. This study, involving three incidents separated by time and location, represents the seminal application of gauze as a sampling media after critical human-shark interactions and strongly supports the practical implementation of these methods in the field.

Opportunities and challenges for microbiomics in ecosystem restoration.

Microbiomics is the science of characterizing microbial community structure, function, and dynamics. It has great potential to advance our understanding of plant-soil-microbe processes and interaction networks which can be applied to improve ecosystem restoration. However, microbiomics may be perceived as complex and the technology is not accessible to all. The opportunities of microbiomics in restoration ecology are considerable, but so are the practical challenges. Applying microbiomics in restoration must move beyond compositional assessments to incorporate tools to study the complexity of ecosystem recovery. Advances in metaomic tools provide unprecedented possibilities to aid restoration interventions. Moreover, complementary non-omic applications, such as microbial inoculants and biopriming, have the potential to improve restoration objectives by enhancing the establishment and health of vegetation communities.

Marine heatwave and reduced light scenarios cause species-specific metabolomic changes in seagrasses under ocean warming.

Climate change and extreme climatic events, such as marine heatwaves (MHWs), are threatening seagrass ecosystems. Metabolomics can be used to gain insight into early stress responses in seagrasses and help to develop targeted management and conservation measures. We used metabolomics to understand the temporal and mechanistic response of leaf metabolism in seagrasses to climate change. Two species, temperate Posidonia australis and tropical Halodule uninervis, were exposed to a combination of future warming, simulated MHW with subsequent recovery period, and light deprivation in a mesocosm experiment. The leaf metabolome of P. australis was altered under MHW exposure at ambient light while H. uninervis was unaffected. Light deprivation impacted both seagrasses, with combined effects of heat and low light causing greater alterations in leaf metabolism. There was no MHW recovery in P. australis. Conversely, the heat-resistant leaf metabolome of H. uninervis showed recovery of sugars and intermediates of the tricarboxylic acid cycle under combined heat and low light exposure, suggesting adaptive strategies to long-term light deprivation. Overall, this research highlights how metabolomics can be used to study the metabolic pathways of seagrasses, identifies early indicators of environmental stress and analyses the effects of environmental factors on plant metabolism and health.

The utility of dust for forensic intelligence: Exploring collection methods and detection limits for environmental DNA, elemental and mineralogical analyses of dust samples.

Environmental DNA (eDNA), elemental and mineralogical analyses of soil have been shown to be specific to their source material, prompting consideration of using the airborne fraction of soil (dust) for forensic intelligence work. Dust is ubiquitous in the environment and is easily transferred to items belonging to a person of interest, making dust analysis an ideal tool in forensic casework. The advent of Massive Parallel Sequencing technologies means metabarcoding of eDNA can uncover bacterial, fungal, and even plant genetic fingerprints in dust particles. Combining this with elemental and mineralogical compositions offers multiple, complementary lines of evidence for tracing the origin of an unknown dust sample. This is particularly pertinent when recovering dust from a person of interest to ascertain where they may have travelled. Prior to proposing dust as a forensic trace material, however, the optimum sampling protocols and detection limits need to be established to place parameters around its utility in this context. We tested several approaches to collecting dust from different materials and determined the lowest quantity of dust that could be analysed for eDNA, elemental composition and mineralogy, whilst still yielding results capable of distinguishing between sites. We found that fungal eDNA profiles could be obtained from multiple sample types and that tape lifts were the optimum collection method for discriminating between sites. We successfully recovered both fungal and bacterial eDNA profiles down to 3 mg of dust (the lowest tested quantity) and recovered elemental and mineralogical compositions for all tested sample quantities. We show that dust can be reliably recovered from different sample types, using different sampling techniques, and that fungi and bacteria, as well as elemental and mineralogical profiles, can be generated from small sample quantities, highlighting the utility of dust for forensic intelligence.

Sulfide intrusion in a habitat forming seagrass can be predicted from relative abundance of sulfur cycling genes in sediments.

Sulfide intrusion from sediments is an increasingly recognized contributor to seagrass declines globally, yet the relationship between sediment microorganisms and sulfide intrusion has received little attention. Here, we use metagenomic sequencing and stable isotope (34S) analysis to examine this relationship in Cockburn Sound, Australia, a seagrass-dominated embayment with a gradient of sulfide stress and seagrass declines. There was a significant positive relationship between sulfide intrusion into seagrasses and sulfate reduction genes in sediment microbial communities, which was greatest at sites with long term seagrass declines. This is the first demonstration of a significant link between sulfur cycling genes present in seagrass sediments and sulfide intrusion in a habitat-forming seagrass that is experiencing long-term shoot density decline. Given that microorganisms respond rapidly to environmental change, the quantitative links established in this study can be used as a potential management tool to enable the prediction of sulfide stress on large habitat forming seagrasses; a global issue expected to worsen with climate change.

Analysis of rapid HIT application to touch DNA samples.

Rapid DNA technology is being utilized for reference profiles worldwide. There is also strong data in the literature to support its use for high-template DNA sources, the same is not true for low-template sources, such as touch DNA; this is a requirement before wider implementation to forensic casework is considered. We report on the Rapid HIT Intel cartridge's ability to facilitate successful amplification of touch DNA to obtain profiles from template deposited on items commonly encountered in forensic casework. Eight items were touched in ten replicates- two were tapelifted, three swabbed, and three directly inserted. Significance was observed in the alleles amplified and RFU with respect to sample type. Three samples performed well: cable tie, fabric, and matchstick. As two of these were directly inserted, this should be considered for any sample small enough. Placement of highly absorbent substrates into the cartridge is not advised as it can cause a lysate-pull error. Heterozygote loci often presented as homozygous (32%-78% loci per profile); this was influenced by substrate type and profile RFU. Loci with larger masses exhibited higher false homozygosity also. Comparison of the donor's profile analyzed was performed against previous datasets analyzing touch DNA through standard workflow, including manual DNA extraction, PCR, and CE separation. These data show that for all substrates, except for a fabric swatch, standard processing is preferential to Rapid HIT analysis. In its current form, rapid DNA technology is not fit for the routine analysis of touch DNA samples in forensic casework.

The influences of dusty environments on the STR typing success of post-detonation touch DNA samples.

As the use of improvised explosive devices (IEDs) in a broad spectrum of offences continues, it is vital that research is performed to assess the capabilities of the forensic DNA profiling technology currently available to provide information as to potential perpetrators. This work investigates some of the most important gaps in our understanding surrounding the poor success rates in DNA profiling obtained through the sampling of touch DNA on post-detonation IED samples. It has been previously suggested that the use of Diamond™ Nucleic Acid Dye may fix cells to a surface, therefore reducing the effect of an experimental process to remove or damage those cells. This was found not to be the case for samples undergoing a detonation as there was no difference in the resultant post-detonation profiles between the stained samples, stained prior to detonation, and unstained samples. The comparison of data from previously performed research, within an enclosed explosives chamber, to real-world outdoor detonation events in a rural and dusty environment was investigated. It was found that there was a significant difference between the environments for the aluminium but not for the battery or electrical tape substrates indicating that environment has the potential to influence STR success through the introduction of PCR inhibitors; humic acid within rural natural dust was introduced here. No difference was observed in cell loss due to the detonation between environments and the dirt within the PCR was higher in the 'outdoor' samples. The effect on cellular retention and damage due to the sample's distance from the charge has been thoroughly investigated through incremental 100 mm exposure. Distance from the charge was found to affect every metric analysed; these being the cell loss from samples, the number of alleles amplified in resultant direct PCR profiles, and the total RFU of the subsequent profiles. These data outline the importance of this work allowing results to be assessed and triage decisions be made accordingly. The analysis of wood, PVC pipe, a mobile phone with rubber buttons, a SIM card, and a circuit board showed that none of these samples at 400 mm from the charge caused substrate specific PCR inhibition. On-site collection teams do not need to triage collection based on these sample types as there was no significant difference observed in their ability to return DNA profiling data. Surface area and inhibitor presence are key variables to consider when determining STR processing workflow for post-detonation samples as for samples with larger surface areas within the outdoor environment PCR post-extraction is preferential to direct PCR.

Exploring tapelifts as a method for dual workflow STR amplification.

Although a version of direct PCR is implemented in forensic laboratories for reference material, its incorporation into workflow for the analysis of touch DNA, as a form of latent DNA, from casework exhibits is not. In addition to concerns about increased sensitivity causing more complex mixtures or the generation of more genetic data implicating an individual superfluous to the context of the alleged event, the complete use of the collected sample in the PCR as template has meant that there is no possibility for data reproducibility when needed. Here it is proposed that the use of tapelifts in touch DNA collection can facilitate replicate direct PCR analysis from a single sample allowing the sample to be re-tested. If all portions of the tapelift result in profiles with allelic and likelihood ratio concordance, these sub-samples may be accepted as technical replicates, thus meeting any accreditation guideline requirements. Furthermore, we assess the use of a single tapelift for both direct PCR and extraction-based PCR workflows to illustrate the potential for benefits of both systems to be facilitated. DNA was deposited by three donors onto six substrates with five sample replicates of each condition. Separation of each tapelift into three portions for three direct PCRs ensued using VeriFiler™ Plus. Separation of single tapelifts into three direct PCRs showed no statistical difference in donor allele calls or RFU, or subsequent LRs associated with their profiles. Comparison of profiles within the single tapelift showed more similarity, with high mixture-to-mixture match likelihoods, than when these sub-samples were compared with profiles generated from other samples. This allows each sub-sample taken from the tapelift to be considered as technical replicates. For dual workflow facilitation assessment, one donor deposited DNA through touch onto six substrates with five research replicates of each. Separation of single tapelifts into two portions, one for direct PCR and the retention and use of the remaining portion for extraction and subsequent PCR, showed no significant difference in allelic yield and subsequent donor comparison LRs. Comparison of deconvoluted profiles produced from a single tapelift showed high mixture-to-mixture match likelihoods, supporting DNA donor concordance. This indicates that removing a portion of a tapelift for direct PCR amplification, while processing the remainder through standard processes, allows increased sensitivity through direct PCR while offering the preparation of an eluate suitable for repeated analyses.

Natural attenuation of legacy hydrocarbon spills in pristine soils is feasible despite difficult environmental conditions in the monsoon tropics.

The Kimberley region of Western Australia is a National Heritage listed region that is internationally recognised for its environmental and cultural significance. However, petroleum spills have been reported at a number of sites across the region, representing an environmental concern. The region is also characterised as having low soil nutrients, high temperatures and monsoonal rain - all of which may limit the potential for natural biodegradation of petroleum. Therefore, this work evaluated the effect of legacy petroleum hydrocarbons on the indigenous soil microbial community (across the domains Archaea, Bacteria and Fungi) at three sites in the Kimberley region. At each site, soil cores were removed from contaminated and control areas and analysed for total petroleum hydrocarbons, soil nutrients, pH and microbial community profiling (using16S rRNA and ITS sequencing on the Illumina MiSeq Platform). The presence of petroleum hydrocarbons decreased microbial diversity across all kingdoms, altered the structure of microbial communities and increased the abundance of putative hydrocarbon degraders (e.g. Mycobacterium, Acremonium, Penicillium, Bjerkandera and Candida). Microbial community shifts from contaminated soils were also associated with an increase in soil nutrients (notably Colwell P and S). Our study highlights the long-term effect of legacy hydrocarbon spills on soil microbial communities and their diversity in remote, infertile monsoonal soils, but also highlights the potential for natural attenuation to occur in these environments.

Cable bacteria at oxygen-releasing roots of aquatic plants: a widespread and diverse plant-microbe association.

Cable bacteria are sulfide-oxidising, filamentous bacteria that reduce toxic sulfide levels, suppress methane emissions and drive nutrient and carbon cycling in sediments. Recently, cable bacteria have been found associated with roots of aquatic plants and rice (Oryza sativa). However, the extent to which cable bacteria are associated with aquatic plants in nature remains unexplored. Using newly generated and public 16S rRNA gene sequence datasets combined with fluorescence in situ hybridisation, we investigated the distribution of cable bacteria around the roots of aquatic plants, encompassing seagrass (including seagrass seedlings), rice, freshwater and saltmarsh plants. Diverse cable bacteria were found associated with roots of 16 out of 28 plant species and at 36 out of 55 investigated sites, across four continents. Plant-associated cable bacteria were confirmed across a variety of ecosystems, including marine coastal environments, estuaries, freshwater streams, isolated pristine lakes and intensive agricultural systems. This pattern indicates that this plant-microbe relationship is globally widespread and neither obligate nor species specific. The occurrence of cable bacteria in plant rhizospheres may be of general importance to vegetation vitality, primary productivity, coastal restoration practices and greenhouse gas balance of rice fields and wetlands.

How many cells are required for successful DNA profiling?

Through advances in fluorescent nucleic acid dye staining and visualisation, targeted collection of cellular material deposited, for example by touch or within a saliva deposit, is possible. In regard to the potential evidentiary value of the deposit the questions remain: 'How many cells are required to generate an informative DNA profile?'; 'How many visualised corneocytes within a touch deposit compared to typical nucleated cells are required in order to achieve successful DNA profiling?'. Diamond TM Nucleic Acid Dye (DD) staining of cellular material, and subsequent visualisation utilising portable fluorescence microscopy, was performed for touch and saliva samples to target defined numbers of cells for collection, by swab and tapelift, and subsequent processing via direct PCR and PCR post-extraction. The resulting DNA quantification data and alleles generated within subsequent DNA profiles could be correlated to the number of cells initially collected to determine cellular threshold requirements for DNA profile generation for each workflow. Full profiles were consistently generated using direct PCR when the template was ≥40 buccal cells collected by either a swab or tapelift. By contrast ≥800 corneocytes collected by swabbing or ≥4,000 corneocytes collected by a tapelift were required to generate same number of STR alleles from touch samples. When samples were processed through a DNA extraction workflow, ≥80 buccal cells were required to generate full profiles from both swab and tapelift, while touch samples required ≥4,000 corneocytes collected by a swab and >8,000 corneocytes collected by a tapelift. The data presented within this study allow for informative sample triage and workflow decisions to be made to optimise STR amplification based on the presence and visual quantification of stained cellular material.

Cutting out the middle clam: lucinid endosymbiotic bacteria are also associated with seagrass roots worldwide.

Seagrasses and lucinid bivalves inhabit highly reduced sediments with elevated sulphide concentrations. Lucinids house symbiotic bacteria (Ca. Thiodiazotropha) capable of oxidising sediment sulphide, and their presence in sediments has been proposed to promote seagrass growth by decreasing otherwise phytotoxic sulphide levels. However, vast and productive seagrass meadows are present in ecosystems where lucinids do not occur. Hence, we hypothesised that seagrasses themselves host these sulphur-oxidising Ca. Thiodiazotropha that could aid their survival when lucinids are absent. We analysed newly generated and publicly available 16S rRNA gene sequences from seagrass roots and sediments across 14 seagrass species and 10 countries and found that persistent and colonising seagrasses across the world harbour sulphur-oxidising Ca. Thiodiazotropha, regardless of the presence of lucinids. We used fluorescence in situ hybridisation to visually confirm the presence of Ca. Thiodiazotropha on roots of Halophila ovalis, a colonising seagrass species with wide geographical, water depth range, and sedimentary sulphide concentrations. We provide the first evidence that Ca. Thiodiazotropha are commonly present on seagrass roots, providing another mechanism for seagrasses to alleviate sulphide stress globally.

Soil microbes of an urban remnant riparian zone have greater potential for N removal than a degraded riparian zone.

Soils in the riparian zone, the interface between terrestrial and aquatic ecosystems, may decrease anthropogenic nitrogen (N) loads to streams through microbial transformations (e.g., denitrification). However, the ecological functioning of riparian zones is often compromised due to degraded conditions (e.g., vegetation clearing). Here we compare the efficacy of an urban remnant and a cleared riparian zone for supporting a putative denitrifying microbial community using 16S rRNA sequencing and quantitative polymerase chain reaction of archaeal and bacterial nitrogen cycling genes. Although we had no direct measure of denitrification rates, we found clear patterns in the microbial communities between the sites. Greater abundance of N-cycling genes was predicted by greater soil ammonium (N-NH4 ), organic phosphorus, and C:N. At the remnant site, we found positive correlations between microbial community composition, which was dominated by putative N oxidisers (Nitrosomonadaceae, Nitrospiraceae and Nitrosotaleaceae), and abundance of ammonia-oxidizing archaea (AOA), nirS, nirK and nosZ, whereas the cleared site had lower abundance of N-oxidisers and N cycling genes. These results were especially profound for the remnant riparian fringe, which suggests that this region maintains suitable soil conditions (via diverse vegetation structure and periodic saturation) to support putative N cyclers, which could amount to higher potential for N removal.

Successful STR amplification of post-blast IED samples by fluorescent visualisation and direct PCR.

Improvised explosive devices (IEDs) present a number of challenges in terms of the generation of forensically relevant information. Inhibition to PCR from sub-optimal sample types as well as from specific substrates has historically meant that extraction prior to PCR has been required. Improvements to STR kit buffers lead to the successful introduction of direct PCR to the analysis of IED-relevant samples, however none of these samples have been exposed to detonations. This study presents data to support the use of direct PCR in the analysis of IED components post-detonation. VeriFiler™ Plus generated informative profiles, containing ≥ 12 autosomal alleles, from samples touched for a maximum of 15 s that were then exposed to a detonation from plastic explosive placed as close as 100 mm. Of the 37 recovered touched items or fragments, 28 contained autosomal alleles from the donor with 18 (49 %) presenting informative profiles that matched the DNA donor. This compared with results following STR PCR post-extraction with one of 11 amplified post-detonation touch DNA samples being informative. The use of Diamond™ Nucleic Acid Dye (DD) staining and visualisation before and after detonation allowed for analysis as to cell loss or damage as a result of the detonation itself and aided in the triaging of samples to be selected for DNA profiling. This is the first record of cellular visualisation and comparison before and after detonation with accompanying STR results on a range of sample types typical of IED constituents. Following comparison of DD visualised cells and STR amplification success, chemical analysis of plastic and electrical tape samples supported substrate-specific inhibition. These data represent the first instance of informative DNA profiles being produced from post-detonation samples using direct PCR, as close as 100 mm from the charge.

Root microbiomes as indicators of seagrass health.

The development of early warning indicators that identify ecosystem stress is a priority for improving ecosystem management. As microbial communities respond rapidly to environmental disturbance, monitoring their composition could prove one such early indicator of environmental stress. We combined 16S rRNA gene sequencing of the seagrass root microbiome of Halophila ovalis with seagrass health metrics (biomass, productivity and Fsulphide) to develop microbial indicators for seagrass condition across the Swan-Canning Estuary and the Leschenault Estuary (south-west Western Australia); the former had experienced an unseasonal rainfall event leading to declines in seagrass health. Microbial indicators detected sites of potential stress that other seagrass health metrics failed to detect. Genera that were more abundant in 'healthy' seagrasses included putative methylotrophic bacteria (e.g. Methylotenera and Methylophaga), iron cycling bacteria (e.g. Deferrisoma and Geothermobacter) and N2 fixing bacteria (e.g. Rhizobium). Conversely, genera that were more abundant in 'stressed' seagrasses were dominated by putative sulphur-cycling bacteria, both sulphide-oxidising (e.g. Candidatus Thiodiazotropha and Candidatus Electrothrix) and sulphate-reducing (e.g. SEEP-SRB1, Desulfomonile and Desulfonema). The sensitivity of the microbial indicators developed here highlights their potential to be further developed for use in adaptive seagrass management, and emphasises their capacity to be effective early warning indicators of stress.

Oxygen loss from seagrass roots coincides with colonisation of sulphide-oxidising cable bacteria and reduces sulphide stress.

Seagrasses thrive in anoxic sediments where sulphide can accumulate to phytotoxic levels. So how do seagrasses persist in this environment? Here, we propose that radial oxygen loss (ROL) from actively growing root tips protects seagrasses from sulphide intrusion not only by abiotically oxidising sulphides in the rhizosphere of young roots, but also by influencing the abundance and spatial distribution of sulphate-reducing and sulphide-oxidising bacteria. We used a novel multifaceted approach combining imaging techniques (confocal fluorescence in situ hybridisation, oxygen planar optodes, and sulphide diffusive gradients in thin films) with microbial community profiling to build a complete picture of the microenvironment of growing roots of the seagrasses Halophila ovalis and Zostera muelleri. ROL was restricted to young root tips, indicating that seagrasses will have limited ability to influence sulphide oxidation in bulk sediments. On the microscale, however, ROL corresponded with decreased abundance of potential sulphate-reducing bacteria and decreased sulphide concentrations in the rhizosphere surrounding young roots. Furthermore, roots leaking oxygen had a higher abundance of sulphide-oxidising cable bacteria; which is the first direct observation of these bacteria on seagrass roots. Thus, ROL may enhance both abiotic and bacterial sulphide oxidation and restrict bacterial sulphide production around vulnerable roots, thereby helping seagrasses to colonise sulphide-rich anoxic sediments.

DNA profiles generated from a range of touched sample types.

Direct PCR from touch DNA has a range of potential applications in the field of forensic investigation for exhibit examination that, under standard extraction methods, rarely produce informative DNA profiles. Previous studies from 'touch DNA' have focussed on fingermarks created under laboratory conditions. Here we report on successful STR DNA profiling from a range of touched items. Direct PCR, with no increase in cycle number, was performed after eight different sample types, typical of those submitted for forensic investigation, were handled by volunteers for a maximum of 15 s to deposit trace amounts of their DNA. Amplifications were performed using either GlobalFiler® or Identifiler® Plus following manufacturer's instructions. These two kits were chosen deliberately as many laboratories worldwide have adopted and validated them in their workflow, thus allowing for direct PCR to be incorporated within their practises easily. It was found that informative STR profiles were obtained from all eight substrates using both STR kits. Identifiler® Plus out-performed GlobalFiler® in terms of the percentage of alleles amplified using the direct PCR approach. Both generated informative profiles from all items and all individuals, at different rates, with Identifiler® Plus being informative in a larger percentage of samples. GlobalFiler® produced profiles with an average of 60% ±â€¯24% (36 ±â€¯15 alleles) alleles present while Identifiler® Plus produced profiles with an average of 96% ±â€¯4% (31 ±â€¯1 alleles) alleles present. A comparison was made between the direct PCR approach and subjecting touched samples to a standard DNA extraction process, both using Identifiler®. An average of 4% of profiles were informative for samples that underwent extraction with 100% being informative from the same subset of samples amplified by direct PCR. Our findings further demonstrate the success of direct PCR to enhance the STR DNA profiles from touch DNA. Further, Identifiler® Plus was found to generate informative profiles more often than GlobalFiler®. Direct PCR is fast, simple, and non-destructive of evidence with the ability to generate informative genetic data where standard methods are likely to fail.