Addressing the challenge of RNA extraction from hard tissues of wild animals is crucial, especially given the species' conservation and the ethical imperative to avoid lethal sampling methods. This study focuses on optimizing a protocol for non-invasive RNA extraction from the caudal scutes of Crocodylus moreletii, an endemic species in the Yucatan Peninsula, Mexico, highlighting the significance of conducting research in tropical areas with limited laboratory access. Accompanying with RNA preservation buffer for the scute tissue, we explored various tissue disruption and homogenization techniques to facilitate RNA isolation and purification. The purity and integrity of RNA were assessed to determine the best extraction method. The optimized protocol involved ultrasonication of 75 mg samples, followed by a 3-hour Proteinase K incubation, yielding RNA with concentrations from 18.7 to 154.7 ng/µL, satisfactory purity (260/280 ratio ∼2), and integrity (RNA Integrity Number >5.5). Further validation through quantitative PCR analyses confirmed the suitability of the extracted RNA for studies on gene expression levels and were sufficient for next-generation sequencing (NGS). This protocol may provide a basis for developing similar methodologies for other non-model species with hard tissues.•This study optimizes non-invasive RNA extraction from crocodile scutes, enabling conservation research and transcriptomic analysis.

Habitat degradation induced by human activities can exacerbate the spread of wildlife disease and could hinder the recovery of imperiled species. The endangered green turtle Chelonia mydas is impacted worldwide by fibropapillomatosis (FP), a neoplastic infectious disease likely triggered by the Scutavirus chelonidalpha5 with coastal anthropogenic stressors acting as cofactors in disease development. Here, we studied fibropapillomatosis dynamics and its demographic consequences using an 11-year capture-mark-recapture dataset in Anse du Bourg d'Arlet/Chaudière (ABAC) and Grande Anse d'Arlet (GA), two juvenile green turtle foraging grounds in Martinique, French West Indies. Afflicted turtles had similar mortality and permanent emigration rates to the non-afflicted ones. Fibropapillomatosis was commonly observed in large individuals and disease recovery may take several years. Consequently, permanent emigration before full recovery from the disease is suspected and might affect the developmental migration success. Additionally, the results revealed that the FP had higher prevalence and severity, and progressed two times faster in ABAC than in GA despite the proximity (< 2 km) and the similarity of the two foraging grounds. The reasons for these differences remain unidentified. Locally, further studies should be focused on the determination of the external and internal cofactors related to the observed FP dynamics. Finally, the investigations should be extended at a global regional scale to determine potential deleterious effect of the FP on the adult life-stage. These perspectives improves upon our overall understanding on the interplay between wildlife diseases, hosts and environmental factors.

Oncogenic viruses play a pivotal role in oncology due to their unique role in unraveling the complexities of cancer development. Understanding the role viruses play in specific cancers is important to provide basic insights into the transformation process, which will help identify potential cellular targets for treatment. This review discusses the diverse role of animal herpesviruses in initiating and promoting various forms of cancer. We will summarize the mechanisms that underlie the development of animal herpesvirus-induced cancer that may provide a basis for developing potential therapeutic interventions or preventative strategies in the future.

The immune system of sea turtles is not completely understood. Sea turtles (as reptiles) bridge a unique evolutionary gap, being ectothermic vertebrates like fish and amphibians and amniotes like birds and mammals. Turtles are ectotherms; thus, their immune system is influenced by environmental conditions like temperature and season. We aim to review the turtle immune system and note what studies have investigated sea turtles and the effect of the environment on the immune response. Turtles rely heavily on the nonspecific innate response rather than the specific adaptive response. Turtles' innate immune effectors include antimicrobial peptides, complement, and nonspecific leukocytes. The antiviral defense is understudied in terms of the diversity of pathogen receptors and interferon function. Turtles also mount adaptive responses to pathogens. Lymphoid structures responsible for lymphocyte activation and maturation are either missing in reptiles or function is affected by season. Turtles are a marker of health for their marine environment, and their immune system is commonly dysregulated because of disease or contaminants. Fibropapillomatosis (FP) is a tumorous disease that afflicts sea turtles and is thought to be caused by a virus and an environmental factor. We aim, by exploring the current understanding of the immune system in turtles, to aid the investigation of environmental factors that contribute to the pathogenesis of this disease and provide options for immunotherapy.

Fibropapillomatosis (FP) is a debilitating tumor disease affecting all species of sea turtles globally. The most probable etiological agent for FP is the chelonid herpesvirus 5 (ChHV5). A 2015-2016 field survey of the sea turtles at Mabul Island, Sabah, Malaysia, found three green turtles (Chelonia mydas) with FP tumors. However, the presence of ChHV5 was confirmed in 7.8% (9/115) green turtles and was absent (0/16) in the hawksbill (Eretmochelys imbricata) turtles, as determined through molecular approaches. Subsequent to this, we managed to conduct field sampling of sea turtles in November 2019, just prior to the pandemic lockdown. Here, we aim to determine the extent of ChHV5 infection, and whether the virus has spread to other species of sea turtles around Mabul Island after the first reports of ChHV5 and FP. A total of 69 tissue samples were obtained from green (63), hawksbill (5), and olive ridley (Lepidochelys olivacea) (1) turtles in November 2019. We observed only one green turtle that exhibited FP tumors. To determine the presence of ChHV5, viral DNA was isolated from all the tissue samples, and polymerase chain reaction (PCR) analysis targeting three highly conserved regions of the virus, i.e., the capsid protein gene, glycoprotein H gene, and glycoprotein B gene, was performed. Out of 63 green turtles, 27 were positive for the presence of the virus. The prevalence of ChHV5 in the green turtles showed an increase of 42.9% as compared to the previous sampling conducted in 2015-2016. Additionally, for the first time, three out of the five hawksbill turtles, and one olive ridley turtle, were also PCR-positive for the virus. In conclusion, this study reveals that there has been an increase in ChHV5 infection among turtles in Mabul Island over the last 3 years. ChHV5 should be considered a potential threat, and mitigation efforts should be taken to prevent the spread of infection among the endangered sea turtles of Mabul Island and surrounding islands within the Coral Triangle.

The green turtle (Chelonia mydas) is a circumglobal species with a wide dietary breadth that varies among regions and life history stages. Comprehensive understanding of foraging ecology over space and time is critical to inform conservation and management of this species and its habitats. Here, we used DNA metabarcoding to test candidate primer sets with 39 gut content homogenates from stranded green turtles (FL, USA) to identify primer sets that maximize detection of food items and specificity of taxonomic classifications. We tested six existing universal primer sets to detect plants, animals, and eukaryotes more broadly (CO1, 18SV1-V3, 18SV4, rbcL, UPA, ITS). The CO1 and 18SV4 primer sets produced the greatest number of dietary amplicon sequence variants (ASVs) and unique taxonomic classifications, and they were the only primer sets to amplify taxa from all three kingdoms relevant to green turtle diet (Animalia, Chromista, and Plantae). Even though the majority of CO1-derived reads were of host origin (>90%), this primer set still produced the largest number of dietary ASVs classified to species among the six primer sets. However, because the CO1 primer set failed to detect both vascular plants and green algae, we do not recommend the use of this primer set on its own to characterize green turtle diet. Instead, our findings support previous research highlighting the utility of using multiple primer sets, specifically targeting CO1 and the V4 region of the 18S gene, as doing so will provide the most comprehensive understanding of green turtle diet. More generally, our results highlight the importance of primer and loci selection and the need to validate primer sets against the study system of interest. The addition of DNA metabarcoding with optimized primer sets to the sea turtle researcher's toolbox will both increase our understanding of foraging ecology and better inform science-based conservation and ecosystem management.

Characterizing polymorphism at the major histocompatibility complex (MHC) genes is key to understanding the vertebrate immune response to disease. Despite being globally afflicted by the infectious tumour disease fibropapillomatosis (FP), immunogenetic variation in sea turtles is minimally explored. We sequenced the α 1 peptide-binding region of MHC class I genes (162 bp) from 268 juvenile green (Chelonia mydas) and 88 loggerhead (Caretta caretta) sea turtles in Florida, USA. We recovered extensive variation (116 alleles) and trans-species polymorphism. Supertyping analysis uncovered three functional MHC supertypes corresponding to the three well-supported clades in the phylogeny. We found significant evidence of positive selection at seven amino acid sites in the class I exon. Random forest modelling and risk ratio analysis of Ch. mydas alleles uncovered one allele weakly associated with smooth FP tumour texture, which may be associated with disease outcome. Our study represents the first characterization of MHC class I diversity in Ch. mydas and the largest sample of sea turtles used to date in any study of adaptive genetic variation, revealing tremendous genetic variation and high adaptive potential to viral pathogen threats. The novel associations we identified between MHC diversity and FP outcomes in sea turtles further highlight the importance of evaluating genetic predictors of disease, including MHC and other functional markers.

Fibropapillomatosis (FP) of sea turtles is characterised by cutaneous tumours and is associated with Chelonid herpesvirus 5 (ChHV5), an alphaherpesvirus from the family Herpesviridae. Here, we provide the first evidence of ChHV5-associated FP in endangered Green turtles (Chelonia mydas) from Sabah, which is located at the northern region of Malaysian Borneo. The aims of our study were firstly, to determine the presence of ChHV5 in both tumour exhibiting and tumour-free turtles using molecular techniques and secondly, to determine the phylogeography of ChHV5 in Sabah. We also aim to provide evidence of ChHV5 infection through histopathological examinations. A total of 115 Green turtles were sampled from Mabul Island, Sabah. We observed three Green turtles that exhibited FP tumours and were positive for ChHV5. In addition, six clinically healthy turtles (with no presence of tumours) were also positive for the virus based on Polymerase Chain Reaction of three viral genes (Capsid protein gene UL18, Glycoprotein H gene UL22, and Glycoprotein B gene UL27). The prevalence of the ChHV5 was 5.22% in asymptomatic Green turtles. Epidermal intranuclear inclusions were identified in tumour lesions upon histopathological examination. In addition, phylogenetic analyses of the UL18, UL22, UL27, and UL30 gene sequences showed a worldwide distribution of the ChHV5 strain with no clear distinction based on geographical location suggesting an interoceanic connection and movement of the sea turtles. Thus, the emergence of ChHV5 in Green turtles in the waters of Sabah could indicate a possible threat to sea turtle populations in the future and requires further monitoring of the populations along the Bornean coast.