Molecular approaches are becoming more prevalent for the diagnosis of neurodegenerative diseases in human medicine and can be extended to diagnosis of wildlife diseases such as chronic wasting disease and other prion diseases. These diseases have been associated with exosome-bound molecular biomarkers of disease progression, such as proteins and micro RNA molecules (miRNA). We tested and optimized a method for exosomal miRNA isolation from minimally invasive, small-volume serum samples obtained from white-tailed deer (Odocoileus virginianus). We confirmed the isolation of exosomes and optimized a commercially available benchtop kit to obtain sufficient and pure RNA for miRNA sequencing. The selected method for RNA extraction combines two 500-μL serum aliquots into one elution column and re-eluting the final product of the column. We identified 137 miRNA present in healthy white-tailed deer that can be used as a baseline to identify putative miRNA biomarkers of disease progression and mechanisms of infection in future comparative disease studies. This approach to biomarker discovery may help to inform biological processes in wildlife populations and provide alternatives to invasive or postmortem samples.

The accessibility to CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) genetic tools has given rise to applications beyond site-directed genome editing for the detection of DNA and RNA. These tools include precise diagnostic detection of human disease pathogens, such as SARS-CoV-2 and Zika virus. Despite the technology being rapid and cost-effective, the use of CRISPR/Cas tools in the surveillance of the causative agents of wildlife diseases has not been prominent. This study presents the development of a minimally invasive, field-applicable and user-friendly CRISPR/Cas-based biosensor for the detection of Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), an infectious disease that has killed more than five million bats in North America since its discovery in 2006. The biosensor assay combines a recombinase polymerase amplification (RPA) step followed by CRISPR/Cas12a nuclease cleavage to detect Pd DNA from bat dermal swab and guano samples. The biosensor had similar detection results when compared to quantitative PCR in distinguishing Pd-positive versus negative field samples. Although bat dermal swabs could be analysed with the biosensor without nucleic acid extraction, DNA extraction was needed when screening guano samples to overcome inhibitors. This assay can be applied to help with more rapid delineation of Pd-positive sites in the field to inform management decisions. With further optimization, this technology has broad translation potential to wildlife disease-associated pathogen detection and monitoring applications.

Although many studies have characterized catarrhine and platyrrhine primate herpesviruses, little is known about herpesviruses in prosimians. We aimed to identify and characterize herpesviruses in prosimians with proliferative lymphocytic disease. DNA was extracted from tissues of 9 gray mouse lemurs (Microcebus murinus) and 3 pygmy slow lorises (Nycticebus pygmaeus) with lymphoproliferative lesions, and we performed nested PCR and sequencing for detection of herpesviruses and polyomaviruses. We identified 3 novel herpesviruses and performed phylogenetic analyses to characterize their relationship with other herpesviruses. A gray mouse lemur herpesvirus clustered with other primate herpesviruses within the subfamily Betaherpesvirinae, just basal to the genus Cytomegalovirus. The other gray mouse lemur herpesvirus and the pygmy slow loris herpesvirus clustered within the subfamily Gammaherpesvirinae, although the relationships within the subfamily were less resolved. Quantitative PCR assays were developed for the 2 new gray mouse lemur viruses, providing specific, faster, less expensive, and quantitative detection tools. Further studies are needed to elucidate the relationship between the presence of these viruses and the severity or presence of lymphoproliferative lesions in prosimians.

The U.S. Geological Survey Community for Data Integration annually supports small projects focusing on data integration for interdisciplinary research, innovative data management, and demonstration of new technologies.This report provides a summary of the 14 projects supported in fiscal year 2019 and outlines their goals, activities, and accomplishments.Proposals in 2019 were encouraged to address the optional disciplinary theme of biosurveillance of emerging invasive species and health threats.

To advance national efforts for the detection and biosurveillance of aquatic invasive species (AIS), we employed a community consensus process to enable the incorporation of environmental DNA (eDNA) detection data into the U.S. Geological Survey's (USGS) Nonindigenous Aquatic Species (NAS) database (https://nas.er. usgs.gov/eDNA/). Our goal was to identify minimum standards and best practices for the verification of eDNA data by working closely with AIS eDNA community practitioners and natural resource managers across government, private and academic sectors. To better inform management decisions, verified AIS eDNA data will be displayed on a separate mapping layer alongside visual sighting data with the inclusion of additional information on the eDNA methods employed to collect and produce the data. To allow for eDNA data display, we produced consensus derived online documents including a submission application and data submission template and are developing a guidance document for detailing the eDNA data submission process. We also developed a communication plan including a mechanism for reporting detections to appropriate managers for consideration prior to display. The products of these efforts are an application and data submission process that will be used in the new environmental DNA data layer on the Nonindigenous Aquatic Species (NAS) database. Herein, we detail how we engaged the eDNA community for consensus of our standards, share lessons learned from the process, and describe the benefits of such an approach at instilling confidence among the research and decision-maker community.

Invasive alien species (IAS) are a rising threat to biodiversity, national security, and regional economies, with impacts in the hundreds of billions of U.S. dollars annually. Proactive or predictive approaches guided by scientific knowledge are essential to keeping pace with growing impacts of invasions under climate change. Although the rapid development of diverse technologies and approaches has produced tools with the potential to greatly accelerate invasion research and management, innovation has far outpaced implementation and coordination. Technological and methodological syntheses are urgently needed to close the growing implementation gap and facilitate interdisciplinary collaboration and synergy among evolving disciplines. A broad review is necessary to demonstrate the utility and relevance of work in diverse fields to generate actionable science for the ongoing invasion crisis. Here, we review such advances in relevant fields including remote sensing, epidemiology, big data analytics, environmental DNA (eDNA) sampling, genomics, and others, and present a generalized framework for distilling existing and emerging data into products for proactive IAS research and management. This integrated workflow provides a pathway for scientists and practitioners in diverse disciplines to contribute to applied invasion biology in a coordinated, synergistic, and scalable manner.

Cold stress syndrome (CSS) in the Florida manatee (Trichechus manatus latirostris) results in perturbations to many physiological pathways, often leading to further illness or death. In this study, we applied a non-targeted lipidomics approach with ultra-high performance liquid chromatography and high-resolution tandem mass spectrometry to characterize changes related to CSS in the lipidomic profiles of manatee plasma. Lipidomic analyses were conducted on healthy manatee (control) and cold-exposed manatee plasma samples with varying concentrations of Serum Amyloid A (SAA), an acute-phase protein that is associated with inflammatory disease. Control manatees (n = 10) were compared to all manatees exposed to cold temperatures (n = 17), and a subset of those manatees with SAA values > 120 μg/mL (n = 9). Increased SAA values were associated with higher levels of various acylcarnitine lipids, while several triacylglycerols and oxidized triacylglycerols were significantly lower in manatees with cold exposure. These identified lipids are critical molecules involved in the maintenance of energy homeostasis and could potentially be examined in conjunction with current physical parameters to characterize cold stress. The ability to detect such differences highlights the addition of lipidomics as a valuable tool in understanding cold stress and potentially other illnesses in manatees. Further investigation into the function of the altered lipids could greatly increase our understanding of lipid metabolism in physiologically stressed manatees as well as other marine mammals and inform future management recovery strategies.

Genome-wide evaluations of genetic diversity and population structure are important for informing management and conservation of trailing-edge populations. North American moose ( Alces alces ) are declining along portions of the southern edge of their range due to disease, species interactions, and marginal habitat, all of which may be exacerbated by climate change. We employed a genotyping by sequencing (GBS) approach in an effort to collect baseline information on the genetic variation of moose inhabiting the species’ southern range periphery in the contiguous United States. We identified 1920 single nucleotide polymorphisms (SNPs) from 155 moose representing three subspecies from five states: A. a. americana (New Hampshire), A. a. andersoni (Minnesota), and A. a. shirasi (Idaho, Montana, and Wyoming). Molecular analyses supported three geographically isolated clusters, congruent with currently recognized subspecies. Additionally, while moderately low genetic diversity was observed, there was little evidence of inbreeding. Results also indicated > 20% shared ancestry proportions between A. a. shirasi samples from northern Montana and A. a. andersoni samples from Minnesota, indicating a putative hybrid zone warranting further investigation. GBS has proven to be a simple and effective method for genome-wide SNP discovery in moose and provides robust data for informing herd management and conservation priorities. With increasing disease, predation, and climate related pressure on range edge moose populations in the United States, the use of SNP data to identify gene flow between subspecies may prove a powerful tool for moose management and recovery, particularly if hybrid moose are more able to adapt.

Bullseye Snakehead Channa marulius (Hamilton 1822) was first detected in the southern Florida town of Tamarac in 2000 and has been expanding its geographic range since. Environmental DNA (eDNA) analysis is a newly-developed technique used to noninvasively detect cryptic or low-density species or those that are logistically difficult-to -study. Genetic material shed into the environment through tissue and body fluids is concentrated from water samples and analyzed for the presence of target species eDNA. To help delineate Bullseye Snakehead's geographic range, we developed and validated a species-specific eDNA assay for both quantitative and droplet digital PCR (ddPCR). We then used ddPCR to assess 16 locations in southeast Florida using 222 water samples collected from 2015 to 2018. Positive eDNA detections were obtained at all six locations that were within the known geographic range of Bullseye Snakehead. Furthermore, eDNA was detected in six of 10 locations that were previously thought to be outside the periphery of the range but hydrologically connected through the extensive canal system. Over the four years of sampling, estimated occurrence rates (psi) remained stable and relatively high (psi = 0.67 [95% credible interval (CI) 0.33-0.95]) near Tamarac, Florida, as compared to the most southern sampling locations (psi = 0.0-0.37). Bullseye Snakehead eDNA estimated occurrence rates in the middle region increased between 2016 (0.28 [95% CI 0.03-0.94]) and 2017 (0.66 [95% CI 0.24-0.98]), potentially reflecting eDNA detections related to a growing or expanding population. Bullseye Snakehead eDNA was detected at low concentrations on the northern and eastern borders of Everglades National Park, which is an important conservation area and UNESCO World Heritage Site. Despite extensive sampling via electrofishing, no Bullseye Snakehead were visually detected in several locations that yielded positive eDNA samples. It is unclear whether eDNA was transported through flowing water or another vector. To date, collection records for this species are confined to urban canals; however, Bullseye Snakehead may use the interconnected system of canals to disperse to natural conservation areas such as Everglades National Park, Big Cypress National Preserve, and Water Conservation Areas, where it may impact native species via predation and competition.

Environmental DNA (eDNA) detection of invasive species can be used to inform management decisions by delimiting occupied ranges and estimating detection probabilities. Environmental DNA is shed into the environment through skin cells and bodily fluids and can be detected in water samples collected from lakes, rivers, and swamps. In south Florida, USA, invasive Burmese pythons (Python bivittatus) occupy much of the Greater Everglades in mostly inaccessible habitat and are credited with causing severe declines in populations of native species. Detection of Burmese pythons by traditional methods, including trapping and visual searching, has been largely ineffective, making eDNA a superior method to identify invaded areas. We adapted a quantitative PCR eDNA assay for use in droplet digital PCR, a state-of-the-art method that improves the precision and accuracy of eDNA detection and quantification. From August 2014 to October 2016, locations in and around Arthur R. Marshall Loxahatchee National Wildlife Refuge in southeast Florida were surveyed for Burmese python eDNA. Positive eDNA detections were made in each of the five sampling events, assessing a total of 399 samples, with moderate-to-high occurrence (Psi=58-91%) and moderate detection (p= 38-70%) probabilities, potentially reduced by a high presence of PCR-inhibiting compounds in the water. The high occurrence rates and geographic distribution of the positive samples within Loxahatchee suggests a steady release of python eDNA from resident Burmese pythons and reduces support for transport of eDNA by boats or flowing water from the north. The first confirmed sighting of a Burmese python in the Refuge occurred in September 2016 after eDNA testing had indicated the presence of pythons. While the established population boundary is thought to be south of Loxahatchee, the eDNA detections indicate a northern range limit at, or north of, the refuge on the eastern side of the Florida peninsula. Our study demonstrates the utility of eDNA for determining more accurate range limits and expansion information for Burmese pythons, and provides a means for assessing control efforts.

To inform management and conservation decisions, environmental DNA (eDNA) methods are used to detect genetic material shed into the water by imperiled and invasive species. Methodological enhancements are needed to reduce filter clogging, PCR inhibition, and false-negative detections when eDNA is at low concentrations. In the first of three simple experiments, we sought to ameliorate filter clogging from particulates and organic material through a scaled-up, multi-filter protocol. We combined four filters in a 5 mL Phenol-Chloroform-Isoamyl (PCI) procedure to allow for larger volumes of water (~1 L) to be filtered rapidly. Increasing the filtered water volume by four times resulted in 4.4X the yield of target DNA. Next, inhibition from organic material can reduce or block eDNA detections in PCR-based assays. To remove inhibitory compounds retained during eDNA isolation, we tested three methods to chemically strip inhibitors from eDNA molecules. The use of CTAB as a short-term (5–8 day) storage buffer, followed by a PCI isolation, resulted in the highest eDNA yields. Finally, as opposed to a linear relationship among increasing concentrations of filtered genomic eDNA, we observed a sharp change between the lower (70–280 ng) and higher (420–560 ng) amounts. This may be important for effectively precipitating eDNA during protocol testing.

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees (Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-c; interleukin (IL)-2, -6, and -10; tumor necrosis factor-a; and the housekeeping genes glyceraldehyde3-phosphate dehydrogenase (GAPDH) and b-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida, US. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-c were significantly higher (P,0.05) in manatees from the east coast of Florida than they were from those from the west coast. We found IL-10 and b-actin to be consistent between sites and identified b-actin as a good candidate for use as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

Cytokines have important roles in the mammalian response to viral and bacterial infections, trauma, and wound healing. Because of early cytokine production after physiologic stresses, the regulation of messenger RNA (mRNA) transcripts can be used to assess immunologic responses before changes in protein production. To detect and assess early immune changes in endangered Florida manatees ( Trichechus manatus latirostris), we developed and validated a panel of quantitative PCR assays to measure mRNA transcription levels for the cytokines interferon (IFN)-γ; interleukin (IL)-2, -6, and -10; tumor necrosis factor-α; and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (reference genes). Assays were successfully validated using blood samples from free-ranging, apparently healthy manatees from the east and west coasts of central Florida, US. No cytokine or housekeeping gene transcription levels were significantly different among age classes or sexes. However, the transcription levels for GAPDH, IL-2, IL-6, and IFN-γ were significantly higher ( P<0.05) in manatees from the east coast of Florida than they were from those from the west coast. We found IL-10 and β-actin to be consistent between sites and identified β-actin as a good candidate for use as a reference gene in future studies. Our assays can aid in the investigation of manatee immune response to physical trauma and novel or ongoing environmental stressors.

We use Tempus blood RNA tubes (Applied Biosystems) during health assessments of American moose (Alces alces spp.) as a minimally invasive means to obtain RNA. Here we describe a novel protocol to additionally isolate high-quality DNA from the supernatant remaining after the RNA isolation methodology. Metrics used to qualify DNA quality included measuring the concentration, obtaining a DNA integrity number from a genomic DNA ScreenTape assay (Agilent), and running the isolated DNA on an agarose gel.

Environmental DNA (eDNA) detection is a technique used to non-invasively detect cryptic, low density, or logistically difficult-to-study species, such as imperiled manatees. For eDNA measurement, genetic material shed into the environment is concentrated from water samples and analyzed for the presence of target species. Cytochrome b quantitative PCR and droplet digital PCR eDNA assays were developed for the 3 Vulnerable manatee species: African, Amazonian, and both subspecies of the West Indian (Florida and Antillean) manatee. Environmental DNA assays can help to delineate manatee habitat ranges, high use areas, and seasonal population changes. To validate the assay, water was analyzed from Florida's east coast containing a high-density manatee population and produced 31 564 DNA molecules l(-1) on average and high occurrence (psi) and detection (p) estimates (psi = 0.84 [0.40-0.99]; p = 0.99 [0.95-1.00]; limit of detection 3 copies mu l(-1)). Similar occupancy estimates were produced in the Florida Panhandle (psi = 0.79 [0.54-0.97]) and Cuba (psi = 0.89 [0.54-1.00]), while occupancy estimates in Cameroon were lower (psi = 0.49 [0.09-0.95]). The eDNA-derived detection estimates were higher than those generated using aerial survey data on the west coast of Florida and may be effective for population monitoring. Subsequent eDNA studies could be particularly useful in locations where manatees are (1) difficult to identify visually (e.g. the Amazon River and Africa), (2) are present in patchy distributions or are on the verge of extinction (e.g. Jamaica, Haiti), and (3) where repatriation efforts are proposed (e.g. Brazil, Guadeloupe). Extension of these eDNA techniques could be applied to other imperiled marine mammal populations such as African and Asian dugongs.

Trichechid herpesvirus 1 (TrHV-1) is currently the only known herpesvirus in any sirenian. We hypothesized that stress may lead to recrudescence of TrHV-1 in manatees, thus making TrHV-1 a potential biomarker of stress. We optimized and validated a TrHV-1 real-time quantitative probe hybridization PCR (qPCR) assay that was used to quantify TrHV-1 in manatee peripheral blood mononuclear cells (PBMCs). Average baseline TrHV-1 loads in a clinically healthy wild Florida manatee (Trichechus manatus latirostris) population (n = 42) were 40.9 ± SD 21.2 copies/100 ng DNA; 19 of 42 manatees were positive. TrHV-1 loads were significantly different between the 2 field seasons (p < 0.025). This optimized and validated qPCR assay may be used as a tool for further research into TrHV-1 in Florida manatees.

A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low-concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species' presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty-indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics.

Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.