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Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Dr. Margaret E. Hunter who is an author on this paper currently serves on the editorial board of the PLOS ONE journal. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

To investigate a Florida manatee (Trichechus manatus latirostris) mortality event following a red tide bloom in Southwest Florida, an RNA sequencing experiment was conducted. Gene expression changes in white blood cells were assessed in manatees rescued from a red tide affected area (n = 4) and a control group (n = 7) using RNA sequencing. The genes with the largest fold changes were compared between the two groups to identify molecular pathways related to cellular and disease processes. In total, 591 genes (false discovery rate <0.05) were differentially expressed in the red tide group. Of these, 158 were upregulated and 433 were downregulated. This suggests major changes in white blood cell composition following an exposure to red tide. The most highly upregulated gene, Osteoclast associated 2C immunoglobulin-like receptor (OSCAR), was upregulated 12-fold. This gene is involved in initiating the immune response and maintaining a role in adaptive and innate immunity. The most highly downregulated gene, Piccolo presynaptic cytomatrix protein (PCLO), was downregulated by a factor of 977-fold. This gene is associated with cognitive functioning and neurotransmitter release. Downregulation of this gene in other studies was associated with neuronal loss and neuron synapse dysfunction. Among the cellular pathways that were most affected, immune response, including inflammation, wounds and injuries, cell proliferation, and apoptosis were the most predominant. The pathway with the most differentially expressed genes was the immune response pathway with 98 genes involved, many of them downregulated. Assessing the changes in gene expression associated with red tide exposure enhances our understanding of manatee immune response to the red tide toxins and will aid in the development of red tide biomarkers., Competing Interests: The authors have declared that no competing interests exist.

The Burmese python (Python bivittatus) is now established as a breeding population throughout south Florida, USA. However, the extent of the invasion, and the ecological impacts of this novel apex predator on animal communities are incompletely known, in large part because Burmese pythons (hereafter "pythons") are extremely cryptic and there has been no efficient way to detect them. Pythons are recently confirmed nest predators of long-legged wading bird breeding colonies (orders Ciconiiformes and Pelecaniformes). Pythons can consume large quantities of prey and may not be recognized as predators by wading birds, therefore they could be a particular threat to colonies. To quantify python occupancy rates at tree islands where wading birds breed, we utilized environmental DNA (eDNA) analysis-a genetic tool which detects shed DNA in water samples and provides high detection probabilities. We fitted multi-scale Bayesian occupancy models to test the prediction that pythons occupy islands with wading bird colonies at higher rates compared to representative control islands containing no breeding birds. Our results suggest that pythons are widely distributed across the central Everglades in proximity to active wading bird colonies. In support of our prediction that pythons are attracted to colonies, site-level python eDNA occupancy rates were higher at wading bird colonies (ψ = 0.88, 95% credible interval [0.59-1.00]) than at the control islands (ψ = 0.42 [0.16-0.80]) in April through June (n = 15 colony-control pairs). We found our water temperature proxy (time of day) to be informative of detection probability, in accordance with other studies demonstrating an effect of temperature on eDNA degradation in occupied samples. Individual sample concentrations ranged from 0.26 to 38.29 copies/μL and we generally detected higher concentrations of python eDNA in colony sites. Continued monitoring of wading bird colonies is warranted to determine the effect pythons are having on populations and investigate putative management activities., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Coauthor MEH is an Academic Editor at PLOS ONE. We confirm that this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models.

In many North American rivers, populations of multiple species of non-native cyprinid fishes are present, including black carp (Mylpharyngodon piceus), grass carp (Ctenopharyngodon idella), bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), common carp (Cyprinus carpio), and goldfish (Carassius auratus). All six of these species are found in the Mississippi River basin and tracking their invasion has proven difficult, particularly where abundance is low. Knowledge of the location of the invasion front is valuable to natural resource managers because future ecological and economic damages can be most effectively prevented when populations are low. To test the accuracy of environmental DNA (eDNA) as an early indicator of species occurrence and relative abundance, we applied eDNA technology to the six non-native cyprinid species putatively present in a 2.6 river mile stretch of the Chicago (IL, USA) canal system that was subsequently treated with piscicide. The proportion of water samples yielding positive detections increased with relative abundance of the six species, as indicated by the number of carcasses recovered after poisoning. New markers for black carp, grass carp, and a common carp/goldfish are reported and details of the marker testing to ensure specificity are provided.