ISOTHERMAL RECOMBINANT POLYMERASE AMPLIFICATION AND CRIPSR(CAS12A) ASSAY DETECTION OF RENIBACTERIUM SALMONINARUM AS AN EXAMPLE FOR WILDLIFE PATHOGEN DETECTION IN ENVIRONMENTAL DNA SAMPLES.

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon (Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum, is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20-40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Effect of pre-harvest sanitizer treatments on Listeria survival, sensory quality and bacterial community dynamics on leafy green vegetables grown under commercial conditions.

Leafy green vegetables (LGVs) have large surface areas and can be colonized by various microorganisms including pathogens. In this study, we investigated the effect of pre-harvest sanitizer treatments on the survival of inoculated proxy pathogen Listeria innocua ATCC 33090 and the natural microbial community of mizuna, rocket (arugula), red chard and spinach grown under commercial conditions. Electrolyzed water (e-water), peracetic acid (PAA), and 1-bromo-3-chloro-5-dimethylhydantoin (BCDMH) were tested against water controls. We also observed the subsequent sensorial changes of harvested, bagged LGV leaves over a period of 12 days within chill storage alongside the growth, diversity and structure of bacterial populations determined using 16S rRNA gene amplicon sequencing and total viable counts (TVC). Treatment with PAA resulted in the highest reductions of L. innocua (2.4-5.5 log units) compared to the other treatments (0.25-2.5 log units). On day 0 (24 h after sanitizer application), the TVC on sanitizer treated LGVs were significantly reduced compared to water controls, except for rocket. During storage at 4.5 (±0.5)°C sanitisers only hindered microbial growth on LGVs initially and did not influence final bacterial population levels, growth rates or changes in LGV sample colour, decay, odour and texture compared to water controls. Shelf-life was not extended nor was it reduced. The community structure on LGV types differed though a core set of bacterial amplicon sequence variants (ASV) were present across all samples. No significant differences were observed in bacterial diversity between sanitizer treatments, however sanitizer treated LGV samples had initially reduced diversity compared to water treated samples. The bacterial compositions observed at the end point of storage considerably differed from what was observed at initial point owing to the increase in abundance of specific bacterial taxa, mainly Pseudomonas spp., the abundance and growth responses differing between LGV types studied. This study provides a better understanding on the microbiology and sensory impact of pre-harvest applied sanitiser treatments on different LGVs destined for commercial food use.

Quantifying impacts of an environmental intervention using environmental DNA.

Environmental laws around the world require some version of an environmental-impact assessment surrounding construction projects and other discrete instances of human development. Information requirements for these assessments vary by jurisdiction, but nearly all require an analysis of the biological elements of ecosystems. Amplicon-sequencing-also called metabarcoding-of environmental DNA (eDNA) has made it possible to sample and amplify the genetic material of many species present in those environments, providing a tractable, powerful, and increasingly common way of doing environmental-impact analysis for development projects. Here, we analyze an 18-month time series of water samples taken before, during, and after two culvert removals in a salmonid-bearing freshwater stream. We also sampled multiple control streams to develop a robust background expectation against which to evaluate the impact of this discrete environmental intervention in the treatment stream. We generate calibrated, quantitative metabarcoding data from amplifying the 12s MiFish mtDNA locus and complementary species-specific quantitative PCR data to yield multispecies estimates of absolute eDNA concentrations across time, creeks, and sampling stations. We then use a linear mixed effects model to reveal patterns of eDNA concentrations over time, and to estimate the effects of the culvert removal on salmonids in the treatment creek. We focus our analysis on four common salmonid species: cutthroat trout (Oncorhynchus clarkii), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka). We find that one culvert in the treatment creek seemed to have no impact while the second culvert had a large impact on fish passage. The construction itself seemed to have only transient effects on salmonid species during the two construction events. In the context of billions of dollars of court-mandated road culvert replacements taking place in Washington State, USA, our results suggest that culvert replacement can be conducted with only minimal impact of construction to key species of management concern. Furthermore, eDNA methods can be an effective and efficient approach for monitoring hundreds of culverts to prioritize culverts that are required to be replaced. More broadly, we demonstrate a rigorous, quantitative method for environmental-impact reporting using eDNA that is widely applicable in environments worldwide.

Signal and noise in metabarcoding data.

Metabarcoding is a powerful molecular tool for simultaneously surveying hundreds to thousands of species from a single sample, underpinning microbiome and environmental DNA (eDNA) methods. Deriving quantitative estimates of underlying biological communities from metabarcoding is critical for enhancing the utility of such approaches for health and conservation. Recent work has demonstrated that correcting for amplification biases in genetic metabarcoding data can yield quantitative estimates of template DNA concentrations. However, a major source of uncertainty in metabarcoding data stems from non-detections across technical PCR replicates where one replicate fails to detect a species observed in other replicates. Such non-detections are a special case of variability among technical replicates in metabarcoding data. While many sampling and amplification processes underlie observed variation in metabarcoding data, understanding the causes of non-detections is an important step in distinguishing signal from noise in metabarcoding studies. Here, we use both simulated and empirical data to 1) suggest how non-detections may arise in metabarcoding data, 2) outline steps to recognize uninformative data in practice, and 3) identify the conditions under which amplicon sequence data can reliably detect underlying biological signals. We show with both simulations and empirical data that, for a given species, the rate of non-detections among technical replicates is a function of both the template DNA concentration and species-specific amplification efficiency. Consequently, we conclude metabarcoding datasets are strongly affected by (1) deterministic amplification biases during PCR and (2) stochastic sampling of amplicons during sequencing-both of which we can model-but also by (3) stochastic sampling of rare molecules prior to PCR, which remains a frontier for quantitative metabarcoding. Our results highlight the importance of estimating species-specific amplification efficiencies and critically evaluating patterns of non-detection in metabarcoding datasets to better distinguish environmental signal from the noise inherent in molecular detections of rare targets.

Toward quantitative metabarcoding.

Amplicon-sequence data from environmental DNA (eDNA) and microbiome studies provide important information for ecology, conservation, management, and health. At present, amplicon-sequencing studies-known also as metabarcoding studies, in which the primary data consist of targeted, amplified fragments of DNA sequenced from many taxa in a mixture-struggle to link genetic observations to the underlying biology in a quantitative way, but many applications require quantitative information about the taxa or systems under scrutiny. As metabarcoding studies proliferate in ecology, it becomes more important to develop ways to make them quantitative to ensure that their conclusions are adequately supported. Here we link previously disparate sets of techniques for making such data quantitative, showing that the underlying polymerase chain reaction mechanism explains the observed patterns of amplicon data in a general way. By modeling the process through which amplicon-sequence data arise, rather than transforming the data post hoc, we show how to estimate the starting DNA proportions from a mixture of many taxa. We illustrate how to calibrate the model using mock communities and apply the approach to simulated data and a series of empirical examples. Our approach opens the door to improve the use of metabarcoding data in a wide range of applications in ecology, public health, and related fields.

Heavy metal wombats? Metal exposure pathways to bare-nosed wombats (Vombatus ursinus) living on remediated tin mine tailings.

Rehabilitation of disused mine sites through stabilisation and botanical restoration is ecologically important, but metal transfer pathways to colonising wildlife are often less understood and have never been studied in marsupials. The rehabilitated Royal George tin mine tailings (Tasmania, Australia) and colonisation by bare-nosed wombats (Vombatus ursinus) represented an opportunity to examine potential metal transfer from mine tailings to an herbivorous marsupial. The aim of this study was to examine metal transfer pathways from the mine tailings to wombats, and to determine if wombats are at risk from metal exposure. Concentrations of metals were measured in the tailings substrate, surface water and vegetation, as well as fur samples from a resident wombat, and non-resident (control) wombats. The mineralogy of the tailings is dominated by quartz, muscovite, feldspars, topaz, kaolinite and calcite. Concentrations of several metals were high (exceeding varying health standards) in the tailings (As, Cu, Hg, Pb, Ni, Zn), water (As, Cd, Cu, Zn) and vegetation (As, Cd, Cu, Pb, Mn, Zn). Relative to non-resident wombats, elevated levels of As, Cd, Cu, Pb and Sn were measured in the fur of a resident wombat. Based on modelling of the exposure pathways, consumption of plant material is the most likely metal transfer pathway for As, Cu and Pb, although the risks from ingestion of tailings to this fossorial marsupial should not be discounted. This study is the first to investigate metal exposure pathways to marsupials using rehabilitated mine tailings. Further research is needed to accurately quantify ecological risks and toxicity for wombats and other marsupials native to mining landscapes.

Microbial biogeography of the wombat gastrointestinal tract.

Most herbivorous mammals have symbiotic microbes living in their gastrointestinal tracts that help with harvesting energy from recalcitrant plant fibre. The bulk of research into these microorganisms has focused on samples collected from faeces, representing the distal region of the gastrointestinal (GI) tract. However, the GI tract in herbivorous mammals is typically long and complex, containing different regions with distinct physico-chemical properties that can structure resident microbial communities. Little work has been done to document GI microbial communities of herbivorous animals at these sites. In this study, we use 16S rRNA gene sequencing to characterize the microbial biogeography along the GI tract in two species of wombats. Specifically, we survey the microbes along four major gut regions (stomach, small intestine, proximal colon, distal colon) in a single bare-nosed wombat (Vombatus ursinus) and a single southern hairy-nosed wombat (Lasiorhinus latifrons). Our preliminary results show that GI microbial communities of wombats are structured by GI region. For both wombat individuals, we observed a trend of increasing microbial diversity from stomach to distal colon. The microbial composition in the first proximal colon region was more similar between wombat species than the corresponding distal colon region in the same species. We found several microbial genera that were differentially abundant between the first proximal colon (putative site for primary plant fermentation) and distal colon regions (which resemble faecal samples). Surprisingly, only 10.6% (98) and 18.8% (206) of amplicon sequence variants (ASVs) were shared between the first proximal colon region and the distal colon region for the bare-nosed and southern hairy-nosed wombat, respectively. These results suggest that microbial communities in the first proximal colon region-the putative site of primary plant fermentation in wombats-are distinct from the distal colon, and that faecal samples may have limitations in capturing the diversity of these communities. While faeces are still a valuable and effective means of characterising the distal colon microbiota, future work seeking to better understand how GI microbiota impact the energy economy of wombats (and potentially other hindgut-fermenting mammals) may need to take gut biogeography into account.

Congenital Diseases in Harbor Seals (Phoca vitulina richardsii) from the Salish Sea.

Postmortem data for harbor seals (Phoca vitulina richardsii) in the Salish Sea were analyzed for epidemiologic trends in congenital diseases. Cleft palate, cleft lips, or both (n=8) and cardiac defects (n=5) were the most common congenital abnormalities, followed by cases with multiple defects (n=4). No temporal trends or spatial clusters of cases were seen from 2003 to 2019, during which time monitoring effort was consistent. Cases could not be linked to specific causes such as environmental contamination or maternal malnutrition. Our study suggests that a yearly prevalence of 2.9%±2.2 is the endemic level of congenital disease in this stable harbor seal population. Continued monitoring of birth defects and overall harbor seal population status could help to identify emerging teratogens.