A minimally invasive, field-applicable CRISPR/Cas biosensor to aid in the detection of Pseudogymnoascus destructans, the causative fungal agent of white-nose syndrome in bats.

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.

A new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong.

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.

Reduction of Paraoxonase Expression Followed by Inactivation across Independent Semiaquatic Mammals Suggests Stepwise Path to Pseudogenization.

Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of protein function; loss of function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages-Pon1 became a pseudogene with genetic lesions, such as stop codons and frameshifts, at least four times independently in aquatic and semiaquatic mammals. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and enzymatic activity across four aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We observe in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding-level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing accumulation of disrupting mutations. The recurrent loss of Pon1 function in aquatic/semiaquatic lineages is consistent with a benefit to Pon1 functional loss in aquatic environments. Accordingly, we examine diving and dietary traits across pinniped species as potential driving forces of Pon1 functional loss. We find that loss is best associated with diving activity and likely results from changes in selective pressures associated with hypoxia and hypoxia-induced inflammation.

When are environmental DNA early detections of invasive species actionable?

Environmental DNA (eDNA) sampling provides sensitive early detection capabilities for recently introduced taxa. However, natural resource managers struggle with how to integrate eDNA results into an early detection rapid response program because positive eDNA detections are not always indicative of an eventual infestation. We used a structured decision making (SDM) framework to evaluate appropriate response actions to hypothetical eDNA early detections of an introduced aquatic plant in Sebago Lake (Maine, USA). The results were juxtaposed to a recent study that used a similar SDM approach to evaluate response actions to hypothetical eDNA early detections of introduced mussels in Jordanelle Reservoir (Utah, USA). We found that eDNA early detections were not actionable in Sebago Lake because the plant's invasion potential was spatially constrained and the current management activities provided acceptable levels of mitigation. In Jordanelle Reservoir, eDNA detections were actionable due to high invasion potential and analyses supported management actions to contain the invasion. The divergent outcomes of the two case studies are related to the unique attributes of the habitats and species, highlighting the utility of the SDM approach when considering an eDNA monitoring program. We use these two case studies to present a general SDM framework and a set of heuristics that can be efficiently applied to eDNA early detection rapid response scenarios and other instances associated with indeterminant eDNA detections, especially when there is an imperative to make decisions as quickly as possible.

Genetic diversity goals and targets have improved, but remain insufficient for clear implementation of the post-2020 global biodiversity framework.

Genetic diversity among and within populations of all species is necessary for people and nature to survive and thrive in a changing world. Over the past three years, commitments for conserving genetic diversity have become more ambitious and specific under the Convention on Biological Diversity's (CBD) draft post-2020 global biodiversity framework (GBF). This Perspective article comments on how goals and targets of the GBF have evolved, the improvements that are still needed, lessons learned from this process, and connections between goals and targets and the actions and reporting that will be needed to maintain, protect, manage and monitor genetic diversity. It is possible and necessary that the GBF strives to maintain genetic diversity within and among populations of all species, to restore genetic connectivity, and to develop national genetic conservation strategies, and to report on these using proposed, feasible indicators.

The first assessment of the genetic diversity and structure of the endangered West Indian manatee in Cuba.

The coastal waters of Cuba are home to a small, endangered population of West Indian manatee, which would benefit from a comprehensive characterization of the population's genetic variation. We conducted the first genetic assessment of Cuban manatees to determine the extent of the population's genetic structure and characterize the neutral genetic diversity among regions within the archipelago. We genotyped 49 manatees at 18 microsatellite loci, a subset of 27 samples on 1703 single nucleotide polymorphisms (SNPs), and sequenced 59 manatees at the mitochondrial control region. The Cuba manatee population had low nuclear (microsatellites HE = 0.44, and SNP HE = 0.29) and mitochondrial genetic diversity (h = 0.068 and π = 0.00025), and displayed moderate departures from random mating (microsatellite FIS = 0.12, SNP FIS = 0.10). Our results suggest that the western portion of the archipelago undergoes periodic exchange of alleles based on the evidence of shared ancestry and low but significant differentiation. The southeast Guantanamo Bay region and the western portion of the archipelago were more differentiated than southwest and northwest manatees. The genetic distinctiveness observed in the southeast supports its recognition as a demographically independent unit for natural resource management regardless of whether it is due to historical isolation or isolation by distance. Estimates of the regional effective population sizes, with the microsatellite and SNP datasets, were small (all Ne < 60). Subsequent analyses using additional samples could better examine how the observed structure is masking simple isolation by distance patterns or whether ecological or biogeographic forces shape genetic patterns.

A framework to integrate innovations in invasion science for proactive management.

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.

Global genetic diversity status and trends: towards a suite of Essential Biodiversity Variables (EBVs) for genetic composition.

Biodiversity underlies ecosystem resilience, ecosystem function, sustainable economies, and human well-being. Understanding how biodiversity sustains ecosystems under anthropogenic stressors and global environmental change will require new ways of deriving and applying biodiversity data. A major challenge is that biodiversity data and knowledge are scattered, biased, collected with numerous methods, and stored in inconsistent ways. The Group on Earth Observations Biodiversity Observation Network (GEO BON) has developed the Essential Biodiversity Variables (EBVs) as fundamental metrics to help aggregate, harmonize, and interpret biodiversity observation data from diverse sources. Mapping and analyzing EBVs can help to evaluate how aspects of biodiversity are distributed geographically and how they change over time. EBVs are also intended to serve as inputs and validation to forecast the status and trends of biodiversity, and to support policy and decision making. Here, we assess the feasibility of implementing Genetic Composition EBVs (Genetic EBVs), which are metrics of within-species genetic variation. We review and bring together numerous areas of the field of genetics and evaluate how each contributes to global and regional genetic biodiversity monitoring with respect to theory, sampling logistics, metadata, archiving, data aggregation, modeling, and technological advances. We propose four Genetic EBVs: (i) Genetic Diversity; (ii) Genetic Differentiation; (iii) Inbreeding; and (iv) Effective Population Size (Ne ). We rank Genetic EBVs according to their relevance, sensitivity to change, generalizability, scalability, feasibility and data availability. We outline the workflow for generating genetic data underlying the Genetic EBVs, and review advances and needs in archiving genetic composition data and metadata. We discuss how Genetic EBVs can be operationalized by visualizing EBVs in space and time across species and by forecasting Genetic EBVs beyond current observations using various modeling approaches. Our review then explores challenges of aggregation, standardization, and costs of operationalizing the Genetic EBVs, as well as future directions and opportunities to maximize their uptake globally in research and policy. The collection, annotation, and availability of genetic data has made major advances in the past decade, each of which contributes to the practical and standardized framework for large-scale genetic observation reporting. Rapid advances in DNA sequencing technology present new opportunities, but also challenges for operationalizing Genetic EBVs for biodiversity monitoring regionally and globally. With these advances, genetic composition monitoring is starting to be integrated into global conservation policy, which can help support the foundation of all biodiversity and species' long-term persistence in the face of environmental change. We conclude with a summary of concrete steps for researchers and policy makers for advancing operationalization of Genetic EBVs. The technical and analytical foundations of Genetic EBVs are well developed, and conservation practitioners should anticipate their increasing application as efforts emerge to scale up genetic biodiversity monitoring regionally and globally.

The founding charter of the Omic Biodiversity Observation Network (Omic BON).

Omic BON is a thematic Biodiversity Observation Network under the Group on Earth Observations Biodiversity Observation Network (GEO BON), focused on coordinating the observation of biomolecules in organisms and the environment. Our founding partners include representatives from national, regional, and global observing systems; standards organizations; and data and sample management infrastructures. By coordinating observing strategies, methods, and data flows, Omic BON will facilitate the co-creation of a global omics meta-observatory to generate actionable knowledge. Here, we present key elements of Omic BON's founding charter and first activities.

Diet composition of the African manatee: Spatial and temporal variation within the Sanaga River Watershed, Cameroon.

The present study aimed to investigate the diet of African manatees in Cameroon to better inform conservation decisions within protected areas. A large knowledge gap on diet and seasonal changes in forage availability limits the ability to develop informed local management plans for the African manatee in much of its range. This research took place in the Sanaga River Watershed, which includes two protected areas in the Littoral Region of Cameroon: the Douala-Edea National Park and the Lake Ossa Wildlife Reserve. We analyzed 113 manatee fecal samples and surveyed shoreline emergent and submerged vegetation within the Sanaga River Watershed. We used microhistological analyses to determine the relative contribution of each plant species to African manatee diets and compared across locations and across seasons (wet vs. dry season). We found that the shoreline vegetation is diverse with over 160 plant species, unevenly distributed across space and season, and dominated by emergent vegetation mostly represented by the antelope grass (Echinochloa pyramidalis). We recorded a total of 36 plant species from fecal samples with a spatial and temporal distribution mostly reflecting that of the corresponding shoreline vegetation. African manatees appear to be primarily opportunistically feeding on available vegetation across the seasons and habitat. This work documents the current, but changing, state of plant availability in the Sanaga River Watershed and reports the African manatee diet in Cameroon for the first time. This information can play a critical role in successfully managing the species and these protected areas. If we wish to protect the African manatee and the aquatic ecosystems within the Sanaga River Watershed, we must understand how forage availability changes over time, especially as its waters become nutrient enriched, eutrophic, and exposed to invasive species of plants in a changing world.

Global Commitments to Conserving and Monitoring Genetic Diversity Are Now Necessary and Feasible.

Global conservation policy and action have largely neglected protecting and monitoring genetic diversity-one of the three main pillars of biodiversity. Genetic diversity (diversity within species) underlies species' adaptation and survival, ecosystem resilience, and societal innovation. The low priority given to genetic diversity has largely been due to knowledge gaps in key areas, including the importance of genetic diversity and the trends in genetic diversity change; the perceived high expense and low availability and the scattered nature of genetic data; and complicated concepts and information that are inaccessible to policymakers. However, numerous recent advances in knowledge, technology, databases, practice, and capacity have now set the stage for better integration of genetic diversity in policy instruments and conservation efforts. We review these developments and explore how they can support improved consideration of genetic diversity in global conservation policy commitments and enable countries to monitor, report on, and take action to maintain or restore genetic diversity.

Opportunities and challenges of macrogenetic studies.

The rapidly emerging field of macrogenetics focuses on analysing publicly accessible genetic datasets from thousands of species to explore large-scale patterns and predictors of intraspecific genetic variation. Facilitated by advances in evolutionary biology, technology, data infrastructure, statistics and open science, macrogenetics addresses core evolutionary hypotheses (such as disentangling environmental and life-history effects on genetic variation) with a global focus. Yet, there are important, often overlooked, limitations to this approach and best practices need to be considered and adopted if macrogenetics is to continue its exciting trajectory and reach its full potential in fields such as biodiversity monitoring and conservation. Here, we review the history of this rapidly growing field, highlight knowledge gaps and future directions, and provide guidelines for further research.

Chronic exposure to glyphosate in Florida manatee.

Florida manatees depend on freshwater environments as a source of drinking water and as warm-water refuges. These freshwater environments are in direct contact with human activities where glyphosate-based herbicides are being used. Glyphosate is the most used herbicide worldwide and it is intensively used in Florida as a sugarcane ripener and to control invasive aquatic plants. The objective of the present study was to determine the concentration of glyphosate and its breakdown product, aminomethylphosphonic acid (AMPA), in Florida manatee plasma and assess their exposure to manatees seeking a warm-water refuge in Crystal River (west central Florida), and in South Florida. We analyzed glyphosate's and AMPA's concentrations in Florida manatee plasma (n = 105) collected during 2009-2019 using HPLC-MS/MS. We sampled eight Florida water bodies between 2019 and 2020, three times a year: before, during and after the sugarcane harvest using grab samples and molecular imprinted passive Polar Organic Chemical Integrative Samplers (MIP-POCIS). Glyphosate was present in 55.8% of the sampled Florida manatees' plasma. The concentration of glyphosate has significantly increased in Florida manatee samples from 2009 until 2019. Glyphosate and AMPA were ubiquitous in water bodies. The concentration of glyphosate and AMPA was higher in South Florida than in Crystal River, particularly before and during the sugarcane harvest when Florida manatees depend on warm water refuges. Based on our results, Florida manatees were chronically exposed to glyphosate and AMPA, during and beyond the glyphosate applications to sugarcane, possibly associated with multiple uses of glyphosate-based herbicides for other crops or to control aquatic weeds. This chronic exposure in Florida water bodies may have consequences for Florida manatees' immune and renal systems which may further be compounded by other environmental exposures such as red tide or cold stress.

Macrogenetic studies must not ignore limitations of genetic markers and scale.

Millette et al. (Ecology Letters, 2020, 23:55-67) reported no consistent worldwide anthropogenic effects on animal genetic diversity using repurposed mitochondrial DNA sequences. We reexamine data from this study, describe genetic marker and scale limitations which might lead to misinterpretations with conservation implications, and provide advice to improve future macrogenetic studies.

Investigating the gene expression profiles of rehabilitated Florida manatees (Trichechus manatus latirostris) following red tide exposure.

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.

Environmental DNA sampling reveals high occupancy rates of invasive Burmese pythons at wading bird breeding aggregations in the central Everglades.

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.

Improving eDNA yield and inhibitor reduction through increased water volumes and multi-filter isolation techniques.

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.

Cytonuclear discordance in the Florida Everglades invasive Burmese python (Python bivittatus) population reveals possible hybridization with the Indian python (P. molurus).

The invasive Burmese python (Python bivittatus) has been reproducing in the Florida Everglades since the 1980s. These giant constrictor snakes have caused a precipitous decline in small mammal populations in southern Florida following escapes or releases from the commercial pet trade. To better understand the invasion pathway and genetic composition of the population, two mitochondrial (mtDNA) loci across 1,398 base pairs were sequenced on 426 snakes and 22 microsatellites were assessed on 389 snakes. Concatenated mtDNA sequences produced six haplotypes with an average nucleotide and haplotype diversity of π = 0.002 and h = 0.097, respectively. Samples collected in Florida from morphologically identified P. bivittatus snakes were similar to published cytochrome oxidase 1 and cytochrome b sequences from both P. bivittatus and Python molurus and were highly divergent (genetic distances of 5.4% and 4.3%, respectively). The average number of microsatellite alleles and expected heterozygosity were N A = 5.50 and H E = 0.60, respectively. Nuclear Bayesian assignment tests supported two genetically distinct groups and an admixed group, not geographically differentiated. The effective population size (N E = 315.1) was lower than expected for a population this large, but reflected the low genetic diversity overall. The patterns of genetic diversity between mtDNA and microsatellites were disparate, indicating nuclear introgression of separate mtDNA lineages corresponding to cytonuclear discordance. The introgression likely occurred prior to the invasion, but genetic information on the native range and commercial trade is needed for verification. Our finding that the Florida python population is comprised of distinct lineages suggests greater standing variation for adaptation and the potential for broader areas of suitable habitat in the invaded range.