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Elusive aquatic wildlife, such as endangered sea turtles, are difficult to monitor and conserve. As novel molecular and genetic technologies develop, it is possible to adapt and optimize them for wildlife conservation. One such technology is environmental (e)DNA – the detection of DNA shed from organisms into their surrounding environments. We developed species‐specific green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle probe‐based qPCR assays, which can detect and quantify sea turtle eDNA in controlled (captive tank water and sand samples) and free ranging (oceanic water samples and nesting beach sand) settings. eDNA detection complemented traditional in‐water sea turtle monitoring by enabling detection even when turtles were not visually observed. Furthermore, we report that high throughput shotgun sequencing of eDNA sand samples enabled sea turtle population genetic studies and pathogen monitoring, demonstrating that noninvasive eDNA techniques are viable and efficient alternatives to biological sampling (e.g., biopsies and blood draws). Genetic information was obtained from sand many hours after nesting events, without having to observe or interact with the target individual. This greatly reduces the sampling stress experienced by nesting mothers and emerging hatchlings, and avoids sacrificing viable eggs for genetic analysis. The detection of pathogens from sand indicates significant potential for increased wildlife disease monitoring capacity and viral variant surveillance. Together, these results demonstrate the potential of eDNA approaches to ultimately help understand and conserve threatened species such as sea turtles. see also the Perspective by Taylor Matthew Wilcox and Mads Reinholdt Jensen [ABSTRACT FROM AUTHOR]

Simple Summary: The Kemp's ridley sea turtle is an endangered species that is susceptible to a tumor disease called fibropapillomatosis (FP) and its associated virus, chelonid alphaherpesvirus 5 (ChHV5). The goal of our study was to describe FP in Kemp's ridley turtles, including estimated disease prevalence and pathologyg, and case demographics and outcomes, to better understand the risk posed by FP to Kemp's ridley population recovery. During 2006–2020, we identified 22 cases of Kemp's ridley turtles with FP, including 12 adult turtles, a reproductively valuable age class. Molecular diagnostics were used to identify ChHV5 DNA in blood (7.8%) and tumor (91.7%) samples collected from free-ranging Kemp's ridley turtles. Genomic sequencing was conducted to identify ChHV5 variants in tumor samples collected from Kemp's ridley turtles with FP. Along with case data, phylogenetic analysis of resultant sequences suggests increasing, spatiotemporal spread of ChHV5 infections and FP among Kemp's ridley turtles in coastal areas, including the Gulf of Mexico and the southwestern Atlantic Ocean, where they share habitat with green sea turtles (in which FP is enzootic). This is concerning because FP has an uncertain pathogenesis, is potentially related to anthropogenic environmental degradation, and can cause suffering and/or death in severely afflicted turtles. Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006–2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation. [ABSTRACT FROM AUTHOR]

Simple Summary: Our research aims to unravel uncertainties relating to the genetic and viral causes of the debilitating sea turtle disease fibropapillomatosis, which affects all seven species of sea turtle. This disease is likely caused by an alphaherpesvirus (ChHV5) and an environmental trigger (e.g., pollution). Fibropapillomatosis is characterised by multiple benign tumours which grow on the skin, eyes and internal organs, and is becoming a threat to sea turtle conservation globally. ChHV5 research is crucial to better provide effective management and conservation of turtles from this disease. This study aimed to compare ChHV5 genomes between geographic regions and sea turtle species and observe how this virus has evolved and changed. ChHV5 genomes harboured differences within and between geographic regions (88–2793 single nucleotide polymorphisms (SNPs) per sequenced genome). Multiple ChHV5 genes were also found to be under varying selective pressures. Phylogenomic and phylogenetic analyses revealed grouping of the virus, mostly by geography rather than by species, and found differences in ChHV5 genomes between tumours from the same individual. This study pioneers the phylogenomic approach to ChHV5 research. This study provides the most comprehensive picture to-date of whole-genome inter-species ChHV5 diversity and provides important baseline ChHV5 genomic data for future comparisons. The spreading global sea turtle fibropapillomatosis (FP) epizootic is threatening some of Earth's ancient reptiles, adding to the plethora of threats faced by these keystone species. Understanding this neoplastic disease and its likely aetiological pathogen, chelonid alphaherpesvirus 5 (ChHV5), is crucial to understand how the disease impacts sea turtle populations and species and the future trajectory of disease incidence. We generated 20 ChHV5 genomes, from three sea turtle species, to better understand the viral variant diversity and gene evolution of this oncogenic virus. We revealed previously underappreciated genetic diversity within this virus (with an average of 2035 single nucleotide polymorphisms (SNPs), 1.54% of the ChHV5 genome) and identified genes under the strongest evolutionary pressure. Furthermore, we investigated the phylogeny of ChHV5 at both genome and gene level, confirming the propensity of the virus to be interspecific, with related variants able to infect multiple sea turtle species. Finally, we revealed unexpected intra-host diversity, with up to 0.15% of the viral genome varying between ChHV5 genomes isolated from different tumours concurrently arising within the same individual. These findings offer important insights into ChHV5 biology and provide genomic resources for this oncogenic virus. [ABSTRACT FROM AUTHOR]