Sophie M. Dupont1,2*
Paco Bustamante2‡
David J. Duffy3,4‡
Jérôme Fort2‡
Guillaume Le Loc’h5‡Pierre Lelong1,6‡
Damien Chevallier1†Mathieu Giraudeau2†- 1BOREA Research Unit, Laboratoire de Biologie des Organismes et des Ecosystèmes Aquatiques, UMR 8067, MNHN, CNRS, SU, IRD 207, UCN, UA, Station de recherche Marine de Martinique, Les Anses d’Arlet, France
- 2Littoral Environnement et Sociétés (LIENSs), UMR 7266, CNRS – La Rochelle Université, La Rochelle, France
- 3Whitney Laboratory for Marine Bioscience and Sea Turtle Hospital, University of Florida, St. Augustine, FL, United States
- 4Department of Biology, University of Florida, Gainesville, FL, United States
- 5IHAP, ENVT, INRAE, Université de Toulouse, Toulouse, France
- 6Université des Antilles, Schoelcher, France
Fibropapillomatosis (FP) is a debilitating tumoral disease affecting sea turtles worldwide. While mainly afflicting immature individuals and potentially altering vital functions, the precise impact of this panzootic on turtle health and survival remains unclear. Moreover, the etiological factors implicated in the FP emergence, development and transmission are not yet definitively identified. Among them, an infection by a spreading herpesvirus and the contamination by pollutants (either organic pollutants and trace elements) are suspected. Here, we provide an overview of discoveries, knowledge and propose hypotheses related to FP within five key FP research areas, i.e., virology studies, transmission studies, contamination studies, host genomic studies, and veterinary treatment assays. Moreover, we recommend urgent research avenues to develop at the interface of virology, epidemiology, ecotoxicology, oncology, physiology, immunology, cellular and evolutionary biology, in order to characterize the dynamics of FP and to predict its consequences on sea turtle populations. Importantly, extending the implementation and development of strong collaborations between rehabilitation centers, field biologists and research laboratories at large geographical scale is required to rapidly increase our knowledge on FP and work towards its effective management.
1 Introduction
Fibropapillomatosis (FP) is a debilitating tumoral disease affecting sea turtles, mainly at the immature stage (Work et al., 2004). It is characterized by single or multiple internal and external fibroepithelial lesions ranging from 0.1 to 30 cm in diameter (Herbst, 1994). Internal lesions can grow on any visceral tissue (Rossi et al., 2021) and are histologically described as fibromas, myxofibromas, and fibrosarcomas (Work et al., 2004). External FP-lesions have also been fairly found on all epithelial soft tissues, as well as on the carapace and plastron (see Figure 1 in Rossi et al., 2016). These external FP-lesions exhibit a variety of colors (black, pink, white) and gross appearances: flat plaques, pedunculated, sessile, verrucous, smooth or polypoid nodules (Page-Karjian et al., 2014; Manes et al., 2023b). Light-microscopy observation of FP-external tumors revealed orthokeratotic hyperkeratosis, epidermal hyperplasia, dermal papillary differentiation, ballooning degeneration of epidermal cells and fibroblastic proliferation in the dermis (see Figure 3 in Reséndiz et al., 2021 for a histological cross section of a skin tumor). Depending on their size, number, position, and degree of invasiveness, these lesions can interfere with vision, feeding and locomotion (Aguirre and Lutz, 2004), potentially leading to deleterious consequences on turtle survival (Work et al., 2004; Chaloupka et al., 2008; Flint et al., 2010a but see Chaloupka et al., 2009; Flint et al., 2010b; Patrício et al., 2011; Hargrove et al., 2016). Numerous molecular and epidemiological pieces of evidence point to a herpesvirus infection as a potential causal agent of FP (Quackenbush et al., 2001; Kang et al., 2008).
The first case of FP was observed in the 1930s in a captive green turtle at the New York Aquarium (Smith and Coates, 1938). Initially, the green sea turtle (Chelonia mydas) was then presumed to be the only species affected by this disease. However, typical FP-(external) lesions have now also been reported in all of the seven sea turtle species. The green turtle still remains the most frequently and severely affected species (see Supplementary Table 1 in Jones et al., 2016 for examples of FP prevalence in all seven sea turtle species) and the disease has reached a panzootic status in this species (see Supplementary Table S1 in Dujon et al., 2021). The full extent of the impact of the FP emergence and spread on population dynamics remains unclear (Jones et al., 2016), but is a topic of prime importance since most sea turtle species are categorized as vulnerable to critically endangered on the IUCN red list (Seminoff, 2023), because of various abiotic and biotic environmental pressures, such as bycatch, vessel strikes, loss of nesting habitat, and climate change (Hamann et al., 2010; Rees et al., 2016).
Attesting the concern of the scientific community for the conservation of sea turtle populations facing this panzootic (Aguirre and Lutz, 2004; Hamann et al., 2010; Rees et al., 2016; Mashkour et al., 2020), a growing number of scientific peer-reviewed papers studying FP have been published over the last two decades, most of them reporting FP-cases in new geographical areas (see a recent example, Origlia et al., 2023) or describing current local coastal FP prevalence (as reviewed in Buenrostro-Silva et al., 2022 for the American continent). Consequently, we now have a better overview of the disease distribution and prevalence over global coastal habitats (but a constant and concomitant effort is still needed to get a clear and updated resolution). However, there is an urgent need for a multidisciplinary, global and standardized approach at the interface of virology, epidemiology, ecotoxicology, oncology, physiology, immunology, cellular and evolutionary biology to characterize the dynamics of FP and predict the consequences of this panzootic on sea turtle health, survival and population dynamics (as mentioned for example in Hargrove et al., 2016; Mashkour et al., 2020; Jones et al., 2022). Here, we propose to apply such an interdisciplinary approach within five key FP research areas: 1) virology studies, 2) transmission studies, 3) contamination studies, 4) host genomic studies, 5) veterinary treatment assays. Finally, this paper then aims A) to present FP-related questions that need to be answered to fill the knowledge gaps and to propose standardized research guidelines and B) to stimulate the development of collaborative research projects in order to help sustain the financial, logistical and technical difficulties associated with the research on this topic.
2 Open questions in FP research
2.1 Do ChHV5 dynamics influence FP development pattern(s) and severity?
Based on the Hill’s disease causation criteria and primary FP transmission studies (Hill, 1965; Herbst et al., 1995, 1996), FP seems to be primarily associated with a herpesvirus infection (Quackenbush et al., 2001; Kang et al., 2008). More specifically, the Chelonid alphaherpesvirus 5 (hereafter designated by its widespread abbreviation ChHV5), is the most plausible viral candidate as it is frequently detected in tumoral tissue from FP-affected individuals (Jacobson et al., 1991; Quackenbush et al., 2001). This fibropapilloma-associated turtle herpesvirus, from the Scutavirus chelonidalpha5 species, is a linear double-stranded deoxyribonucleic acid (DNA) virus from the subfamily Alphaherpesvirinae (Benkő et al., 2021). At this time, four ChHV5 variants have been identified in marine turtles: the eastern Pacific, western Atlantic/eastern Caribbean, mid-west Pacific and Atlantic variants (Patrício et al., 2012). Variants are geographic in nature, not being specific to different sea turtle species or rates of disease presentation (Work et al., 2020; Whitmore et al., 2021; Farrell et al., 2022). However, while the viral origin of FP might explain the rapid spread of the disease worldwide (Work et al., 2015b), its implication in the development and dynamics needs to be (molecularly) confirmed. Indeed, on one hand, this virus has already been successfully detected in FP-afflicted turtles (using molecular and microscopy techniques, Quackenbush et al., 2001 and Jacobson et al., 1991 respectively) and also cultured in vitro with turtle tumoral cells and organotypic skin culture (Work et al., 2017). But, on the other hand, only three of the four Koch’s postulates (Rivers, 1937) have been fulfilled so far (Work et al., 2009). Specifically, the isolation of ChHV5 from FP-afflicted turtle and its in vitro culture using standard cell monolayers have not been successfully achieved yet, while a wide variety of validated protocols used to induce viral replication has been tested (i.e., treatment with diverse chemical modulators of replication, incubation at varying temperatures, co-cultivation with known-infected fibroblasts, Work et al., 2009; 2020). Moreover, similar to many herpesviruses, ChHV5 has the capacity to enter a state of latency after a lytic infection (Page-Karjian et al., 2017; Farrell et al., 2021). The difficulty to confirm a causal link between ChHV5 infection and tumor development is also associated with the fact that ChHV5 is near ubiquitous in sea turtle populations. Indeed, absence of FP lesions does not necessarily imply an absence of ChHV5 infection (Page-Karjian et al., 2015; Alfaro-Núñez et al., 2016). Healthy tissue samples from both FP-afflicted and clinically healthy turtles can carry ChHV5 DNA and ribonucleic acid (RNA) (Quackenbush et al., 2001; Farrell et al., 2022). Consequently, the prevalence of individuals positive for ChHV5 is generally much higher than the prevalence of individuals with FP lesions. This observation can be explained by several factors. Firstly, cases of turtle tumor regression have been documented (Guimarães et al., 2013; Manes et al., 2023b), implying that turtles might be tested positive for the virus without showing any tumor-related symptoms. Secondly, ChHV5 has been detected in populations which have never been reported to be afflicted by FP tumors (such as Mediterranean sea turtles, Serra et al., 2023), thus ChHV5 infection alone does not seem sufficient to lead to the development of FP tumors. Thirdly, as proposed by the “viral hit and run hypothesis” (Ambinder, 2000; Niller et al., 2011), if the virus is the key initiator, its action likely happens very early in the oncogenesis process (lytic phase) and the virus might no longer be needed for tumor growth (latent phase, Farrell et al., 2021). Limited information is currently available on how the dynamics of these lytic and latent states of ChHV5 influence the emergence, growth and regression of FP tumors: A) increased viral load in high tumor burdens is due to latent viral DNA replication, rather than increased lytic activity and B) new grown tumors do not show an increase in ChHV5 expression (a proxy for lytic viral infection) compared to established tumors (Farrell et al., 2021; Yetsko et al., 2021). Finally, the development of FP lesions might also depend on the infected tissue and/or the local viral load and potentially only occurs when a certain threshold is locally reached (Jones et al., 2016; Duffy and Martindale, 2019). It should also be noted that using viral metagenomic methodologies, at least 3 additional viruses have been proposed as potential etiological agents of FP in sea turtles: the Dyozetapapillomavirus 1 (Siddell et al., 2020), also named Chelonia mydas papillomavirus 1 – CmPV1 (Mashkour et al., 2021), the Chelonid alphaherpesvirus 6 – ChHV6 (Page-Karjian et al., 2020a), and the sea turtle tornovirus – STTV1 (Ng et al., 2009). However, few little data is currently available regarding their implication into FP pathogenosis. To note, CmPV1 seems to be an especially relevant FP-associated pathogen candidate as papillomaviruses are oncogenic at a much lower load than herpesviruses, and this virus has been detected at non-negligeable prevalence in FP-affected individuals (Mashkour et al., 2018).
Based on this literature review, we strongly recommend further research to confirm the implication of a viral pathogen in FP development, and to identify the etiological agent(s) causing this pathology and its (their) role(s) in triggering FP tumor emergence, development, and spread.
First, with methodological and technical improvements, the fulfilment of the fourth Koch’s postulate will definitively confirm the implication of ChHV5 in FP etiology (Work et al., 2009). This approach should also need to be considered for CmPV1 to assess its etiological implications. In this line, conducting cell culture experiments where cells collected from healthy individuals are exposed to fresh tumor extracts containing the virus(es) should be one of the main future priorities (see successful method described in Work et al., 2017 for ChHV5, and in Mashkour et al., 2018 for CmPV1). Importantly, inoculation should now be done using pure virus, but it then requires to develop laboratory techniques allowing both culture and isolation of ChHV5 and CmPV1. Moreover, to be as informative as possible, an experimental setup is also expected to include transmission electron microscopy of infected cells. Work et al. (2017) developed an innovative cell culture set up to culture ChHV5, however, it was not readily scalable to get cell-free ChHV5 at a level that could be used to fulfill Koch’s postulates. Therefore, prior to ChHV5 inoculation experiments, this ChHV5 cell culture model would need to be refined further or alternative scalable models to be established. Same process should now be initiated for CmPV1.
While exploration of ChHV5 genome has already been initiated (Ackermann et al., 2012; Morrison et al., 2018; Whitmore et al., 2021), new studies of the viral genomes are needed as the complete ChHV5 reference genome remains incomplete, with a (suspected repetitive) region still not sequenced. In this line, novel long-read sequencing technologies (e.g., Pacific Biosciences or Oxford Nanopore Technologies) are ideally placed to complete the full reference genome of ChHV5. Considering the few publication already available, sequencing global ChHV5 genomes should help glean useful information to now robustly identify and confirm the virulence factors involved in the FP context. Assuming that the FP viral origin is true, such an approach will also be particularly appropriate to understand the dynamics of clinical symptoms stages (i.e., from clinically healthy to the development of tumors until complete recovery or death).
Characterizing the temporal viral dynamics (latent vs lytic) in relation to clinical symptoms should also be achieved. To do so, detailed transcriptomic and immunohistochemical profiling of various tumors should be achieved (see the methodology used on external new, established and postsurgical regrown tumors and internal lung and kidney tumors by Farrell et al., 2021; Yetsko et al., 2021). Notably, running these analyses for a wide range of external tumors should help determine whether the viral activity differs according to the size, colour and characteristics of the tumors as well as their anatomical position. Extending this approach should also help characterize the tumors and understand why they present such high phenotypical diversity and variety in growth rate (Manes et al., 2023b). In addition, increasing the number of individuals tested should participate in clarifying how the FP-associated differential gene expression varies according to the individual-specific clinical symptoms stage (i.e., viral infection but clinically healthy, tumor emergence, tumor growth, tumor regression, complete recovery, death). Finally, drawing the transcriptomic and immunohistochemical profiles of virus-infected but not FP-affected individuals (i.e., clinically health individuals) should be conducted. Such studies will help complete the description of the viral dynamics for every clinical symptom stage and specifically highlight the implication of the viral etiological candidates in FP emergence, which seems to actually be the most sensitive stage to the viral infection (Farrell et al., 2021). To go further, the use of innovative molecular histological approaches (such as RNAscope) should also be implemented to describe turtle cellular modifications associated with the viral etiological candidates and then characterize their modes of action.
To note, the lack of consensus on the protocols used to detect and quantify the two main viruses potentially associated with FP makes it difficult to compare data obtained by different studies (see differences in ChHV5 detection using quantitative Polymerase Chain Reaction (PCR), nested-PCR, or singleplex PCR techniques, Alfaro-Núñez and Gilbert, 2014; Jones et al., 2020). This methodological bias may then explain the contradictory results found in the current literature (Jones et al., 2020). Therefore, in the future, we strongly recommend the use of the validated laboratory techniques for qPCR, RNA-seq and DNA-seq, regarding both DNA and RNA extraction as well as viral detection and quantification (Page-Karjian et al., 2015; Blackburn et al., 2021; Mashkour et al., 2021; Yetsko et al., 2021; Farrell et al., 2022). Importantly, we do not seek here to be proscriptive about which ChHV5 qPCR assay should be adopted as the global standard. Ideally, the decision which assay(s) to adopt should come from consensus building across the international FP research community. In our opinion, the subject of standardization should be the topic of a specific workshop e.g., at an international sea turtle conference, and based on a collaborative interlaboratory comparison of method performance conducted altogether by global FP researchers (see the conclusive study conducted for SARS-CoV-2 molecular detection, Deng et al., 2022). In our laboratories, both the Page-Karjian et al. (2015) UL30 and Mashkour et al. (2021) Dpol qPCR assays, which hit somewhat overlapping regions of the same ChHV5 gene, have worked well (Atlantic, Caribbean and Gulf of Mexico samples). The Mashkour et al. (2021) Dpol assay was originally utilized for Australian samples, indicating it would likely function well in global populations. This laboratory protocol standardization will allow the generation of comparable, consistent and reliable datasets at the global scale. Moreover, we also recommend to use standardized tissue and tumor sample collection procedures (sterile biopsy punch of 4 to 6 mm) and storage practices (RNA later or simply frozen at −80°C for DNA analyses). As far as possible, we also recommend to homogenize the samples collected as well as to collect paired samples (FP tumors and nearby healthy tissue). Importantly, we recommend to preferentially collect tissue samples rather than blood samples which have significantly lower ChHV5 loads even in heavy FP-afflicted turtles (Page-Karjian et al., 2015). Moreover, varying ChHV5 detection between fresher and historical samples has been reported by a number of laboratories, for sea turtle tissues and blood samples, and marine leech samples. Consequently, since ChHV5 may be degraded even at −80°C (Kelley, 2022), we encourage to perform analyses within one year after sample collection to avoid false negatives measurements.
2.2 What are the main drivers of FP epidemiology?
Fibropapillomatosis was first observed on a captive green turtle (Smith and Coates, 1938). Since then, FP has been increasingly reported in wild neritic turtles (no pelagic turtle with FP has been recorded so far for logistical constraints), and has been classified as an epizootic disease since the late 1980s (Dujon et al., 2021). Concerningly, the disease is reported in all major ocean basins occupied by sea turtles (Herbst, 1994) with drastic differences in prevalence at both spatial and temporal scales (see for instance local annual FP prevalence in Shaver et al., 2019; Jones et al., 2022; Roost et al., 2022). Several non-exclusive hypotheses have been proposed to explain the spreading of the FP causative agent(s) in turtle populations. Body fluids, tumor shedding into water, or physical contacts might be some routes of transmission in addition to external parasites, and especially Ozobranchus leeches, as disease vectors (Greenblatt et al., 2004; Work et al., 2015b; Farrell et al., 2021; Roost et al., 2022). Interestingly, individuals from sympatric turtle species in a given geographical area usually host the same viral variant, suggesting a viral horizontal transmission (Herbst et al., 2004; Ene et al., 2005; Greenblatt et al., 2005; Patrício et al., 2012; Rodenbusch et al., 2014; Ariel et al., 2017; Jones et al., 2020). To note, a vertical transmission from mother to offspring also seems to be plausible as ChHV5 has been detected in both hatchling tissue samples and crawl tracks from tumor free hatchlings (Farrell et al., 2021, 2022).
Ethical considerations make in vivo experimental approaches not desirable to study FP transmission (although successful experimental inoculations have been conducted, as described in Herbst et al., 1995). However, ex vivo experimental approaches may not be robust enough to infer transmission modalities. Therefore, as the next step in transmission studies, we encourage to focus on viral phylodynamics, eDNA and histological studies, as complementary avenues of research to reveal information on transmission dynamics. To do so, in-depth genomic characterization of the virus should be pursued across broader geographic areas to understand how the viral agent circulates among individuals from a given population and worldwide (Patrício et al., 2012; Ariel et al., 2017; Whitmore et al., 2021). Moreover, it will help identify how the different variants of ChHV5 vary in prevalence at both local and large geographical scales. Finally, phylogenomics will improve our understanding of transmission ability (see promising results for the moneypox virus, Yu et al., 2023, and the SARS-CoV-2, Turakhia et al., 2022), which is crucial to understand the observed variations in disease prevalence. As shedding from tumors appears to be a probable route of transmission (Work et al., 2014; Farrell et al., 2021), it is now crucial to determine the persistency of the virus (ChHV5 and others) in the environment by monitoring viral agent shedding in water from turtle rehabilitated tanks. To validate the vertical transmission hypothesis, detection and quantification of ChHV5 in hatchlings at nest emergence, using swabs, tissues from deceased hatchlings, egg shells and membrane as well as eDNA approaches from the crawl tracks of emerging hatchlings, should be conducted (Farrell et al., 2021, 2022). Such non-invasive approaches will help determine if hatchlings have already been exposed to ChHV5 by the time they are leaving the nest (without indicating exactly at which point from conception to leaving the nest that infection is occurring), a crucial information for the development of effective mitigation measures while minimizing impacts of invasive studies on these endangered species. Finally, the influence of environmental stressors (population density, diet, pathogen pressure) and individual physiological status (immune status, stress levels) should also be determined, using advanced computational models accounting for confounding factors, to understand when individuals are more susceptible to both viral transmission and viral activity. It will help characterize the time and intensity of the early immune response mounted by the host, as it is currently not understood why the early host immune response is not sufficient to prevent tumor development and growth (Yetsko et al., 2021). Specifically, we recommend the routine measurement of white blood cell count and the determination of associated heterophil/lymphocyte ratio (H/L ratio) at the global geographical scale. Indeed, these parameters are among the easiest, cheapest, most repeatable and informative blood immune markers to collect (see detailed methodology in Kophamel et al., 2022).
2.3 How may pollutants trigger FP development and dynamics?
Fibropapillomatosis physiopathology is suspected to be multifactorial, with environmental abiotic and biotic variables affecting the growth dynamics of FP lesions (Herbst and Klein, 1995; Aguirre and Lutz, 2004; Foley et al., 2005; Work et al., 2015a; Dujon et al., 2021; Yetsko et al., 2021). This postulate is supported by the high differences in FP prevalence across both global and local geographical scales (Roost et al., 2022; Vanstreels et al., 2023). Exposure to immunomodulatory environmental stressors might enable ChHV5, a relatively benign opportunistic pathogen, to reach a certain viral load and cross its oncogenic threshold (Duffy and Martindale, 2019). However, such relationships between environmental triggers, turtle immunosuppression and ChHV5 infection have not been confirmed in the FP context so far (Yetsko et al., 2021). Among the abiotic and biotic potential candidates, contaminants are regularly proposed as plausible ones because of their potential influence on both the viral dynamics as well as the host physiological status and immune response (Aguirre et al., 1994). Specifically, contamination with organic pollutants and metallic trace elements are considered as a major turtle threat as they are commonly found in oceans worldwide, can accumulate in organs (Brodie et al., 2014) and have already been associated with turtle decreased health conditions (chlorinated organic pollutants, Camacho et al., 2013; trace elements, Perrault et al., 2017). In line with this, higher FP coastal prevalence have recurrently been found in juvenile and sub-adult individuals residing in anthropized areas with contaminated waters such as near-shore waters and lagoons close to high intensity agricultural, industrial and urban areas (Van Houtan et al., 2014). However, while some studies reported significant associations between FP prevalence or severity and levels of PCBs (Yan et al., 2018), and trace elements (da Silva et al., 2016; Perrault et al., 2017); others found no association (either with organic pollutants, Keller et al., 2014; Sánchez-Sarmiento et al., 2017; or trace elements, Pérez et al., 2023).
To increase our understanding of these potential associations between environmental contaminant and FP dynamics, we first encourage to standardize the methodology to measure pollutant levels, i.e., from tissue sampling to the analytical methods applied, for generating comparable data worldwide. Indeed, reported contrasting results may be explained by the different tissues (blood, heart, kidneys, liver, muscle, carapace, fat, eggs) used to measure contaminant levels: each of which has varying (lipidic) composition and therefore different bioaccumulation properties (e.g., the liver exhibits high (lipidic) bioaccumulation properties while blood has low ones, see Brodie et al., 2014). We recommend to use blood and keratinized tissues, two different but common sample matrices in ecotoxicological studies. First, it will provide insights into both the current and accumulated contamination experienced by any individual, respectively (Schneider et al., 2015). The choice of these two sample matrices is also motivated by the fact that 1) they are of limited invasiveness and can then be collected on live individuals (after obtaining appropriate ethical approbation) and 2) laboratory protocols already exist to measure numerous organic pollutants and trace elements levels on these matrices. Finally, sample processing should be standardized and contamination measurements should be conducted following appropriate laboratory techniques validated for the molecule to detect, and approved from consensus among the global FP research community. In terms of research directions, we first propose studying contamination from both FP-afflicted and healthy turtles living in the same area. Secondly, concomitant measurements of contaminant residue levels and viral presence and load between healthy and FP-afflicted turtles across several seasons (detailed in Jones et al., 2016) should also be pursued. Importantly, rather than searching for particular contaminants, future studies should implement the studied chemicals list based on local water contamination reports. Indeed, the effect of contaminants on organisms depends on their bioavailability and mixture toxicity effects resulting from chemicals’ synergetic and antagonistic modes of action (cocktail effects, Benejam et al., 2010), as well as on the entire abiotic and biotic context of the studied ecosystem (see Rattner and Heath, 2002 for a review of the abiotic factors affecting contaminant toxicity). In other words, a given chemical should be identified as crucial for FP development in a given area but not in another. Thus, finding a unique or a given cocktail of contaminants associated with FP at the worldwide scale might be unlikely. However, by identifying contaminants locally associated with FP emergence and/or development, we might be able to highlight the pollutant chemical properties and modes of action playing a crucial role in FP development and to target them in other geographical regions. Ultimately, by determining the chemical features which could explain the relationship between turtle contamination and FP prevalence, we should then be able to establish whether such association is mediated by the efficiency of turtle immune system. Finally, conducting a global meta-analysis considering detailed standardized studies across multiple locations should provide an useful tool for disentangling some of the mixed ecotoxicological narrative complexity associated with FP (Dujon et al., 2021; Manes et al., 2023a).
2.4 How do host genomics alterations influence the FP development?
The majority of tumor types, whether initially induced by pathogens, environmental exposures or chance mutation, are ultimately driven by alterations in the host genome and transcriptome within tumor cells (Hanahan and Weinberg, 2011). Such oncogenic mutations can come in a number of forms, from small single nucleotide variants to largescale chromosomal rearrangements. These mutations can inactivate genes (such as tumor suppressors), alter gene functioning, or increase gene expression levels, ultimately transforming cells into cancer cells by promoting rapid cell proliferation, and evasion of programmed cell death and immune system controls. Additionally, epigenetic changes can also induce the activation of oncogenic signaling pathways and the inhibition of inherent tumor suppressor defense mechanisms. Despite the prominent role of mutations and oncogenic signaling pathways in driving tumor formation and development, they were historically understudied in the FP context [e.g., tumor whole genome sequencing (WGS/genomic DNA) has only been employed for a small amount of tumor (Yetsko et al., 2021)], with the majority of research effort generally placed on viral investigations. Failing to consider host dynamics adequately has hampered our understanding of this enigmatic disease, slowing the development of effective mitigation, treatment, and prevention strategies. However, in recent years, an increasing number of studies have begun to investigate changes in host FP biology, from immune-related changes, mutational burdens and targetable perturbations to the signaling pathways driving tumor growth (Blackburn et al., 2021; Perrault et al., 2021; Yetsko et al., 2021).
The application of omics technologies, particularly those honed for human cancer research, such as transcriptomics – functionally validated in cell lines, animal models and clinical trials, are enabling the rapid elucidation of the underlying host molecular mechanistic drivers of FP, as well as the identification of diagnostic, prognostic and drug targets. As a striking evidence, the first ever sea turtle FP transcriptomics study contributed to the establishment of a post-surgical drug treatment which cut FP eye tumor regrowth rates from 67% down to 18% (Duffy and Martindale, 2019). Moreover, the few available transcriptomics studies of FP tumor tissue compared with non-tumor tissue have revealed a core oncogenic signaling network driving tumor growth identical to human pan-cancer drivers (Duffy et al., 2018; Yetsko et al., 2021). There are broad transcriptional differences between FP external tumors, and less common internal tumors. However, the oncogenic signaling pathways: mitogen-activated protein kinase (MAPK), Wingless and Int-1 (Wnt), transforming growth factor beta (TGFβ) and tumor necrosis factor (TNF) are common to both of them (Yetsko et al., 2021) and may in the future be therapeutically targeted to expand treatment options. Specifically, these FP transcriptomic signaling changes most related to patient outcome are in tumor suppressor pathways, particularly apoptotic and immune response genes. Concomitantly, sea turtles which retained higher expression of these tumor suppressor genes within their tumors had better rehabilitation outcomes (Blackburn et al., 2021). In addition to revealing viral dynamics, novel treatment options, and serving as putative rehabilitation outcome biomarkers, transcriptomic and genomic profiling of FP tumors can help identify likely contaminants contributing to FP tumorigenesis (Yetsko et al., 2021). Indeed, for other cancers, applying deep genomic sequencing to tumors revealed environmental contaminants responsible for initiating tumorigenesis (COSMIC, 2018; Stammnitz et al., 2018). Similarly, transcriptomic profiles associated with exposure to organic pollutants, trace elements, and viruses were detected in FP tumors (Yetsko et al., 2021). Such approaches should then be pursued to complement field-based environmental contaminant, and blood/tissue burden contaminant studies.
Fibropapillomatosis tumors do harbor mutations within their cancer cell genomes (Yetsko et al., 2021). To improve our fundamental understanding of the nature of FP, further research should be conducted, in larger cohorts of turtles, examining the specific mutational events driving FP tumor formation and growth. Such research can also identify shared mutational FP drivers between tumors and individuals, elucidate anti-cancer treatment options and identify DNA mutational signatures of tumor initiating environmental exposures. Epigenetic modifications have been shown to play a prominent role in many human cancer types, but epigenetic analysis has yet to be applied to FP tumor biology (Hanahan and Weinberg, 2011). FP epigenetics should be investigated as a matter of urgency, as it will likely also lead to novel insights into this panzootic disease.
Building on the outcome of gene expression prognostic biomarker discovery, with putative FP biomarkers already identified (Yetsko et al., 2021), the development of RT-qPCR assay could enable to find rapid, cost-effective gene expression-based host biomarkers (i.e., RNA) for applications in rehabilitation and field settings. Similarly, whole genome sequencing can identify recurrent FP-associated mutations. Of the identified mutations, some are likely to have prognostic value, as is the case with most human cancers. Therefore, qPCR assays could also be developed as prognostic biomarkers, targeting host genomic mutations (gDNA). When used at scale, qPCR or RT-qPCR can cost less than $10 USD per sample. Such outcome biomarkers could help quantify the severity of FP for free-roaming populations. Additionally, they could help inform likely outcomes, and treatment and management decisions within rehabilitation facilities. Indeed, it could contribute to classify patients into those likely to respond to intervention, and those whose tumors are too aggressive, as aggressive and responsive tumors have different underlying molecular profiles, i.e., gene expression profiles and mutational burdens. Host biomarkers for the presence of internal tumors should be particularly beneficial for early detection in rehabilitating sea turtles, as well as for providing a survey tool for monitoring the prevalence of internal tumors in wild populations. The growing awareness of the centrality of host immune and genomic alterations to the development, progression and outcome of FP will help globally tackle this panzootic.
2.5 How to increase treatment efficiency and prophylaxis for FP-affected turtles?
Wild FP-afflicted turtles found lethargic and floating on the sea surface or washed up on the shore are generally temporarily admitted in local rehabilitation facilities (when existing) to be cured before being released back (Manire et al., 2017). The decision to euthanize FP-afflicted turtles is justified, in most regions, by the presence of internal tumors. Indeed, these are currently incurable through medication, and surgical removal of internal tumors is not routinely conducted because it requires specific technical expertise. In addition it is only possible for few internal tumors locations due to the turtle hard shell. However, diagnosis of internal tumors currently requires the appropriate veterinary equipment, i.e., a computed tomography (CT) scanner or X-ray, which is rarely present in rehabilitation facilities due to financial limitations (but see Li et al., 2022 for a promising platform based on ChHV5 glycoprotein B detection). Most often, the clinical cares provided to FP-affected turtles are the surgical removal of external tumors (using CO2 lasers notably, Raiti, 2008) and the treatment of secondary infections (Page-Karjian et al., 2014). However, tumor regrowth has been reported in at least 50% of the surgically treated turtles that have been released and subsequently re-stranded (Farrell et al., 2018; Page-Karjian et al., 2019). Such observation shows that the complete removal of FP tumors is not always possible and rehabilitation success (i.e., survival probability and release success) may be correlated to tumor burden for the majority of FP turtles (Stacy et al., 2019). While one could then wonder whether releasing a few cured individuals each year should have positive and significant effects on local population dynamics, there have been documented cases of previously FP-treated turtles reaching sexual maturity and successfully nesting, thereby contributing to future generations. Importantly, considering the viral FP origin as true, the non-negligible proportion of tumor regrowth should indicate that tumor-free individuals were still carrying out ChHV5 DNA. Actually, sea turtles with higher ChHV5 transcriptional loads (RNA-seq) had the best rehabilitation outcomes (Farrell et al., 2021). In other words, individuals with more active virus fared better than those with lower levels of active virus. This indicates that lytic ChHV5 virus may actually improve the likelihood of turtle survival, possibly by initiating a stronger host immune response (unlike latent virus evading the host immune system, Krump and You, 2018), which could then complement surgical tumor removal by better targeting tumors (Blackburn et al., 2021; Farrell et al., 2021). In the same sea turtle patient cohort, higher expression levels of immune-related host genes were also associated with better patient outcomes (Yetsko et al., 2021). Then, the appropriate research avenue to explore is now determining whether releasing ChHV5-positive tumor-free turtles back into their native contaminated and degraded habitats will not likely lead to re-occurrence of the disease, compromising their rehabilitation success.
With the constant progress of the veterinary medicine, new therapeutic approaches to treat these turtles are expected, with the objective to increase survival rate and reduce tumor reoccurrence. Interestingly, with the recent trend of applying advanced human oncology knowledge to wildlife medicine (Duffy and Martindale, 2019), there is hope of developing new clinical treatment in a decent timeframe. Such reasoning is particularly applicable in the FP context as similarities between human and turtle tumoral cells have been documented (Duffy et al., 2018; Yetsko et al., 2021). This approach is one of the most promising as there is already a panel of efficient orally deliverable drugs used in human oncology that may be administrated routinely in rehabilitation centers after their validation through pharmacokinetic and toxicology studies, i.e., immunomodulators or anti-cancer drugs such as Vismodegib, targeting host genetic perturbations through identified oncogenic signaling pathways, like SHH and Wnt pathways (but see also MAPK and TGF signaling, Duffy et al., 2018; Yetsko et al., 2021); or anti-cancer drugs, such as 5-Fluorouracil or Bleomycin acting as cytotoxic agents (Donnelly et al., 2019). To note, 5-Fluorouracil has already shown promising results with a significant reduction of post-surgical eye FP tumor regrowth, from 67% down to 18% (Duffy et al., 2018). Lysine may also be administrated to prevent tumor regrowth when conducting surgically FP lesions removal (Page-Karjian et al., 2014). Finally, any attempts to use anti-viral drugs for FP treatment, targeting specifically herpesvirus, such as Acyclovir or Ganciclovir, have failed so far, having no effect (D. J. Duffy, unpublished data). Such observation would corroborate with the “viral hit and run” hypothesis which implies that anti-viral treatment may only be efficient during the very early FP stages. However, when turtles are admitted in rehabilitation centers, tumors are already self-sufficient replicating due to oncogenic mutations and transcriptional changes in the host genome and transcriptome, with any remaining virus which may largely be latent. Promisingly, at the beginning of 2024, the first autogenous vaccine therapy for chelonid herpesvirus has successfully been applied in a juvenile green turtle in Colombia (Castro et al., 2024). While this finding opens a new era for FP treatment, it is important to keep in mind that there is a crucial lack of hindsight on such treatment in wild animals (see notably the case studies of avian cholera in albatross species and west Nile virus in the California condor). Then, before considering vaccination as an appropriate preventive cure, several questions must be answered. Notably, which individuals to target (clinically healthy individuals randomly captured in the wild vs FP-affected turtles from rehabilitation centers)? How frequently does the vaccine need to be administrated per individual? Is there any window of efficiency (hatchlings vs immature turtles vs breeding males and females)? Which proportion of the population to vaccine in order to ensure an efficient protection of the whole population?
The most important point to develop, related to improving treatments, is the implementation of strong collaborations between rehabilitation centers and research laboratories worldwide. Indeed, rehabilitated turtles are a unique opportunity to increase our knowledge on FP. Notably, they are regularly checked (at least once a week) which is essential in the FP context as tumors can double in size in less than two weeks (Farrell et al., 2018). Moreover, the entire removal of external tumors – as part of the therapeutic process – allows us to collect significant quantity of tumor tissue that could be used to assess oncogenic and viral dynamics according to tumor size, color, and aspect as well as to their status (regrowth, new growth, established). Moreover, this ready sample access can help follow up as well as descriptively and functionally validate detected correlations between environmental cofactors and FP. Such close collaboration between rehabilitation centers and research laboratories has already provided us numerous crucial insights on the dynamics of the potential viral etiological agents in American FP-afflicted turtles (see for instance Duffy et al., 2018; Page-Karjian et al., 2019, 2020b, 2021; Blackburn et al., 2021; Farrell et al., 2021; Yetsko et al., 2021). It should now be globally extended to confirm these local results and draw general conclusions. Moreover, the regular health and condition record of FP-afflicted turtles should help us monitor the turtle immune status all along the recovery process (from their transfer in rehabilitation center to their release or death). This should be determinant in understanding how the viral agents interfere with host immune function, especially in the context of tumoral regrowth and new growth but also when tumor regression is observed. Such research issue is feasible by weekly monitoring selected health blood parameters (hematocrit, H/L ratio, glycemia, potassium) and comparing external aspect of turtle post-tumor removal through standardized pictures. This methodology will also provide a chance to identify secondary infections and opportunistic pathogens (Work et al., 2003), more likely due to their FP-related immunosuppression (Work et al., 2001; Page-Karjian et al., 2014). Moreover, by monitoring them after release in the wild through photo-identification (i.e., a non-invasive, cheap and standardized method to study FP prevalence, Hancock et al., 2023), it should be possible to precisely evaluate the probability of tumor development as well as their survival probability. Finally, such FP-afflicted turtles should be ideal patients for any veterinary treatment assay to conduct in the next years.
3 Conclusion
Fibropapillomatosis is a complex neoplastic multifactorial disease. As highlighted throughout this article, numerous and crucial knowledge gaps need to be filled, to increase our understanding of this disease. Specifically, the interplay between environmental triggers (e.g., pollutants), viral triggers (e.g., ChHV5) and host responses (e.g., immune response, gene expression changes and mutational burdens) requires further elucidation and studies focusing on the functional understand of all these three aspects and their interrelatedness, rather than attempting to examine them in isolation, should be prioritized. Several of the approaches proposed here, such as blood chemistry (e.g., white blood counts and H/L ratio), genomics and transcriptomics assays (e.g., qPCR and RT-qPCR-assay respectively) as well as veterinary treatment trials, require time but also high logistic and financial supports, limiting their advancement. Because budgets allocated worldwide to FP studies are currently quite limited, increasing local and global collaborative projects should help share costs to reach our scientific objectives in a reasonable timeframe. Finally, it is now essential to standardize the protocols used, by choosing robust and validated methods available in the literature (i.e., viral detection, tumor scoring, sampling, storage and matrix processing) to achieve powerful comparative studies at the global scale. As an ultimate step, it will contribute to improve treatment protocols and to propose appropriate conservation measures at the local scale (Mashkour et al., 2020; Jones et al., 2022).
Author contributions
SMD: Writing – original draft, Writing – review & editing. PB: Writing – review & editing. DJD: Writing – original draft, Writing – review & editing. JF: Writing – review & editing. GLL: Writing – review & editing. PL: Writing – review & editing. DC: Funding acquisition, Project administration, Writing – review & editing. MG: Supervision, Writing – original draft, Writing – review & editing.
Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the Fonds Européen de Développement Régional PO/FEDER/FSE 2014-2020 – Collectivité Territoriale de Martinique (Convention n° MQ0017449), the Direction de l'Environnement, de l'Aménagement et du Logement (DEAL) Martinique, France (Convention N°2017/164894), the Office de l′Eau (ODE) Martinique, France (Convention n°180126) and the Office Français de la Biodiversité (OFB, Parc Naturel Marin de Martinique).
Acknowledgments
We thank all the people around the world who are devoting to the rehabilitation and preservation of sea turtles afflicted by Fibropapillomatosis. PB is an honorary member of the IUF (Institut Universitaire de France).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
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References
Ackermann M., Koriabine M., Hartmann-Fritsch F., de Jong P. J., Lewis T. D., Schetle N., et al. (2012). The genome of chelonid herpesvirus 5 harbors atypical genes. PloS One 7, e46623. doi: 10.1371/journal.pone.0046623
Aguirre A. A., Balazs G. H., Zimmerman B., Galey F. D. (1994). Organic contaminants and trace metals in the tissues of green turtles (Chelonia mydas) afflicted with fibropapillomas in the Hawaiian Islands. Mar. pollut. Bull. 28, 109–114. doi: 10.1016/0025-326X(94)90547-9
Aguirre A. A., Lutz P. L. (2004). Marine turtles as sentinels of ecosystem health: Is fibropapillomatosis an indicator? EcoHealth 1, 275–283. doi: 10.1007/s10393-004-0097-3
Alfaro-Núñez A., Bojesen A. M., Bertelsen M. F., Wales N., Balazs G. H., Gilbert M. T. P. (2016). Further evidence of Chelonid herpesvirus 5 (ChHV5) latency: High levels of ChHV5 DNA detected in clinically healthy marine turtles. PeerJ 4, e2274. doi: 10.7717/peerj.2274
Alfaro-Núñez A., Gilbert M. T. P. (2014). Validation of a sensitive PCR assay for the detection of Chelonid fibropapilloma-associated herpesvirus in latent turtle infections. J. Virol. Methods 206, 38–41. doi: 10.1016/j.jviromet.2014.05.019
Ambinder R. F. (2000). Gammaherpesviruses and “Hit-and-run”. oncogenesis Am. J. Pathol. 156, 1–3. doi: 10.1016/S0002-9440(10)64697-4
Ariel E., Nainu F., Jones K., Juntunen K., Bell I., Gaston J., et al. (2017). Phylogenetic variation of Chelonid Alphaherpesvirus 5 (ChHV5) in populations of green turtles Chelonia mydas along the Queensland Coast, Australia. J. Aquat. Anim. Health 29, 150–157. doi: 10.1080/08997659.2017.1330783
Benejam L., Benito J., García-Berthou E. (2010). Decreases in condition and fecundity of freshwater fishes in a highly polluted reservoir. Water Air Soil Poll. 210, 231–242. doi: 10.1007/s11270-009-0245-z
Benkő M., Brandt C. R., Bryant N. A., Dastjerdi A., Davison A. J., Depledge D. P., et al. (2021)Abolish 6 species and rename 1 family, 4 genera and 124 species in the order Herpesvirales. Available online at: https://ictv.global/taxonomy/taxondetails?taxnode_id=202201423.
Blackburn N. B., Leandro A. C., Nahvi N., Devlin M. A., Leandro M., Martinez Escobedo I., et al. (2021). Transcriptomic profiling of fibropapillomatosis in green sea turtles (Chelonia mydas) from South Texas. Front. Immunol. 12. doi: 10.3389/fimmu.2021.630988
Brodie J., Ariel E., Thomas C., O’Brien D., Berry K. (2014). Links between water quality and marine turtle health. TropWATER—Tropical Water Aquat. Ecosystem Res. 88.
Buenrostro-Silva A., García-Grajales J., Sánchez-Nava P., Ruiz-Gómez M. D. L. (2022). What do we know about sea turtle fibropapillomatosis studies in the American continent? A bibliographic review. Lat. Am. J. Aquat. Res. 50, 343–353. doi: 10.3856/vol50-issue3-fulltext-2787
Camacho M., Luzardo O. P., Boada L. D., Jurado L. F. L., Medina M., Zumbado M., et al. (2013). Potential adverse health effects of persistent organic pollutants on sea turtles: Evidences from a cross-sectional study on Cape Verde loggerhead sea turtles. Sci.Total Environ. 458, 283–289. doi: 10.1016/j.scitotenv.2013.04.043
Castro L. R., Villalba-Viscaíno V., Oviedo Á., Zambrano E., Dávila A., Naranjo G., et al. (2024). Case report: Diagnosis and autogenous vaccine treatment of herpesvirus in a green turtle (Chelonia mydas) in Santa Marta, Colombia. Front. Vet. Sci. 11. doi: 10.3389/fvets.2024.1258209
Chaloupka M., Balazs G. H., Work T. M. (2009). Rise and fall over 26 years of a marine epizootic in Hawaiian green sea turtles. J. Wildl. Dis. 45, 1138–1142. doi: 10.7589/0090-3558-45.4.1138
Chaloupka M., Work T. M., Balazs G. H., Murakawa S. K. K., Morris R. (2008). Cause-specific temporal and spatial trends in green sea turtle strandings in the Hawaiian Archipelago, (1982–2003). Mar. Biol. 154, 887–898. doi: 10.1007/s00227-008-0981-4
COSMIC (2018) COSMIC signatures of mutational processes in human cancer. Available online at: http://cancer.sanger.ac.uk/cosmic/signatures.
da Silva C. C., Klein R. D., Barcarolli I. F., Bianchini A. (2016). Metal contamination as a possible etiology of fibropapillomatosis in juvenile female green sea turtles Chelonia mydas from the southern Atlantic Ocean. Aquat. Toxicol. 170, 42–51. doi: 10.1016/j.aquatox.2015.11.007
Deng K., Uhlig S., Goodman L. B., Ip H. S., Killian M. L., Nemser S. M., et al. (2022). Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States. J. Vet. Diagn. Invest. 34, 825–834. doi: 10.1177/10406387221115702
Donnelly K. A., Papich M. G., Zirkelbach B., Norton T. M., Szivek A., Burkhalter B., et al. (2019). Plasma bleomycin concentrations during electrochemotherapeutic treatment of fibropapillomas in green turtles Chelonia mydas. J. Aquat. Anim. Health 31, 186–192. doi: 10.1002/aah.10067
Duffy D. J., Martindale M. Q. (2019). Perspectives on the expansion of human precision oncology and genomic approaches to sea turtle fibropapillomatosis. Commun. Biol. 2, 54. doi: 10.1038/s42003-019-0301-1
Duffy D. J., Schnitzler C., Karpinski L., Thomas R., Whilde J., Eastman C., et al. (2018). Sea turtle fibropapilloma tumors share genomic drivers and therapeutic vulnerabilities with human cancers. Commun. Biol. 1, 63. doi: 10.1038/s42003-018-0059-x
Dujon A. M., Schofield G., Venegas R. M., Thomas F., Ujvari B. (2021). Sea turtles in the cancer risk landscape: A global meta-analysis of fibropapillomatosis prevalence and associated risk factors. Pathogens 10, 1295. doi: 10.3390/pathogens10101295
Ene A., Su M., Lemaire S., Rose C., Schaff S., Moretti R., et al. (2005). Distribution of chelonid fibropapillomatosis-associated herpesvirus variants in Florida: molecular genetic evidence for infection of turtles following recruitment to neritic developmental habitats. J. Wildl. Dis. 41, 489–497. doi: 10.7589/0090-3558-41.3.489
Farrell J. A., Rachel T., Martindale M. Q., Duffy D. J. (2018). Characterisation of fibropapillomatosis tumour growth profiles in green sea turtles (Chelonia mydas). Testudo 8, 12–29.
Farrell J. A., Whitmore L., Mashkour N., Rollinson-Ramia D., Thomas R. S., Eastman C. B., et al. (2022). Detection and population genomics of sea turtle species via noninvasive environmental DNA analysis of nesting beach sand tracks and oceanic water. Mol. Ecol. Resour. 22, 2471–2493. doi: 10.1111/1755-0998.13617
Farrell J. A., Yetsko K., Whitmore L., Whilde J., Eastman C. B., Rollinson-Ramia D., et al. (2021). Environmental DNA monitoring of oncogenic viral shedding and genomic profiling of sea turtle fibropapillomatosis reveals unusual viral dynamics. Commun. Biol. 4, 565. doi: 10.1038/s42003-021-02085-2
Flint M., Limpus C. J., Patterson-Kane J. C., Murray P. J., Mills P. C. (2010a). Corneal fibropapillomatosis in green sea turtles (Chelonia mydas) in Australia. J. Comp. Pathol. 142, 341–346. doi: 10.1016/j.jcpa.2009.10.012
Flint M., Patterson-Kane J. C., Limpus C. J., Mills P. C. (2010b). Health surveillance of stranded green turtles in Southern Queensland, Australia 2006–2009): an epidemiological analysis of causes of disease and mortality. EcoHealth 7, 135–145. doi: 10.1007/s10393-010-0300-7
Foley A. M., Schroeder B. A., Redlow A. E., Fick-Child K. J., Teas W. G. (2005). Fibropapillomatosis in stranded green turtles (Chelonia mydas) from the eastern United States, (1980-98): Trends and associations with environmental factors. J. Wildl. Dis. 41, 29–41. doi: 10.7589/0090-3558-41.1.29
Greenblatt R. J., Work T. M., Balazs G. H., Sutton C. A., Casey R. N., Casey J. W. (2004). The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas). Virol. 321, 101–110. doi: 10.1016/j.virol.2003.12.026
Greenblatt R. J., Work T. M., Dutton P., Sutton C. A., Spraker T. R., Casey R. N., et al. (2005). Geographic variation in marine turtle fibropapillomatosis. J. Zoo Wildlife Med. 36, 527–530. doi: 10.1638/04-051.1
Guimarães S. M., Gitirana H. M., Wanderley A. V., Monteiro-Neto C., Lobo-Hajdu G. (2013). Evidence of regression of fibropapillomas in juvenile green turtles Chelonia mydas caught in Niterói, southeast Brazil. Dis. Aquat. Org. 102, 243–247. doi: 10.3354/dao02542
Hamann M., Godfrey M. H., Seminoff J. A., Arthur K., Barata P. C. R., Bjorndal K. A., et al. (2010). Global research priorities for sea turtles: Informing management and conservation in the 21st century. Endanger. Species Res. 11, 245–269. doi: 10.3354/esr00279
Hanahan D., Weinberg R. A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646–674. doi: 10.1016/j.cell.2011.02.013
Hancock J. M., Choma J., Mainye L., Wambi P., Stelfox M. R., Polyak M. M., et al. (2023). Using Photo-ID to document and monitor the prevalence of fibropapilloma tumours in a foraging aggregation of green turtles. Front. Mar. Sci. 10. doi: 10.3389/fmars.2023.1217683
Hargrove S. A., Work T. M., Brunson S., Foley A. M., Balazs G. H., Girard A., et al. (2016). “Proceedings of the 2015 international summit on fibropapillomatosis: global status, trends, and population impacts. U.S. Dep. Commer., NOAA Tech. Memo,” in NOAA-TM-NMFS-PIFSC-054, 1–85. doi: 10.7289/V5/TM-PIFSC-54
Herbst L. H. (1994). Fibropapillomatosis of marine turtles. Annu. Rev. Fish Dis. 4, 389–425. doi: 10.1016/0959-8030(94)90037-X
Herbst L. H., Ene A., Su M., Desalle R., Lenz J. (2004). Tumor outbreaks in marine turtles are not due to recent herpesvirus mutations. Curr. Biol. 14, R697–R699. doi: 10.1016/j.cub.2004.08.040
Herbst L. H., Jacobson E. R., Moretti R., Brown T., Sundberg J. P., Klein P. A. (1995). Experimental transmission of green turtle fibropapillomatosis using cell-free tumor extracts. Dis. Aquat. Org. 22, 1–12. doi: 10.3354/dao022001
Herbst L., Klein P. (1995). Green turtle fibropapillomatosis: challenges to assessing the role of environmental cofactors. Environ. Health Perspect. 103, 27–30. doi: 10.1289/ehp.95103s427
Herbst L. H., Moretti R., Brown T., Klein P. A. (1996). Sensitivity of the transmissible green turtle fibropapillomatosis agent to chloroform and ultracentrifugation conditions. Dis. Aquat. Org. 25, 225–228. doi: 10.3354/dao025225
Hill A. B. (1965). The environment and disease: Association or causation. Proc. R. Soc Med. 58, 295–300. doi: 10.1177/003591576505800503
Jacobson E. R., Buergelt C., Williams B., Harris R. K. (1991). Herpesvirus in cutaneous fibropapillomas of the green turtle Chelonia mydas. Dis. Aquat. Org. 12, 1–6. doi: 10.3354/dao012001
Jones K., Ariel E., Burgess G. W., Read M. (2016). A review of fibropapillomatosis in green turtles (Chelonia mydas). Vet. J. 212, 48–57. doi: 10.1016/j.tvjl.2015.10.041
Jones K., Burgess G., Budd A. M., Huerlimann R., Mashkour N., Ariel E. (2020). Molecular evidence for horizontal transmission of chelonid alphaherpesvirus 5 at green turtle (Chelonia mydas) foraging grounds in Queensland, Australia. PloS One 15, e0227268. doi: 10.1371/journal.pone.0227268
Jones K., Limpus C. J., Brodie J., Jones R., Read M., Shum E., et al. (2022). Spatial distribution of fibropapillomatosis in green turtles along the Queensland coast and an investigation into the influence of water quality on prevalence. Conserv. Sci. Pract. 4, e12755. doi: 10.1111/csp2.12755
Kang K. I., Torres-Velez F. J., Zhang J., Moore P. A., Moore D. P., Rivera S., et al. (2008). Localization of fibropapilloma-associated herpesvirus in green turtles (Chelonia mydas) by in-situ hybridization. J. Comp. Pathol. 139, 218–225. doi: 10.1016/j.jcpa.2008.07.003
Keller J. M., Balazs G. H., Nielsen F., Rice M., Work T. M., Jensen B. A. (2014). Investigating the potential role of persistent organic pollutants in Hawaiian green sea turtle fibropapillomatosis. Environ. Sci. Technol. 48, 7807–7816. doi: 10.1021/es5014054
Kelley J. (2022). Fibropapillomatosis and Chelonid herpesvirus 5 dynamics in juvenile green turtles of the Indian River Lagoon, Florida, USA. Master thesis (Orlando, FL: University of Central Florida). Available at: https://stars.library.ucf.edu/etd2020/1031.
Kophamel S., Rudd D., Ward L. C., Shum E., Ariel E., Mendez D., et al. (2022). Haematological and biochemical reference intervals for wild green turtles (Chelonia mydas): a Bayesian approach for small sample sizes. Conserv. Physiol. 10, coac043. doi: 10.1093/conphys/coac043
Krump N. A., You J. (2018). Molecular mechanisms of viral oncogenesis in humans. Nat. Rev. Microbiol. 16, 684–698. doi: 10.1038/s41579-018-0064-6
Li T.-H., Hsu W.-L., Chen C.-Y., Chen Y.-C., Wang Y.-C., Tsai M.-A., et al. (2022). Preparation of recombinant glycoprotein B (gB) of Chelonid herpesvirus 5 (ChHV5) for antibody production and its application for infection detection in sea turtles. Sci. Rep. 12, 11022. doi: 10.1038/s41598-022-15281-9
Manes C., Carthy R. R., Hull V. (2023a). A coupled human and natural systems framework to characterize emerging infectious diseases—the case of fibropapillomatosis in marine turtles. Animals 13, 1441. doi: 10.3390/ani13091441
Manes C., Herren R. M., Page-Karjian A., Dunlap F. D., Skibicki C. A., Rollinson-Ramia D., et al. (2023b). Green turtle fibropapillomatosis: Tumor morphology and growth rate in a rehabilitation setting. Vet. Sci. 10, 421. doi: 10.3390/vetsci10070421
Manire C. A., Norton T. M., Stacy B. A., Innis C. J., Harms C. A. (2017). Sea turtle health and rehabilitation. J. Ross Publishing Plantation Florida p, 1045.
Mashkour N., Jones K., Kophamel S., Hipolito T., Ahasan S., Walker G., et al. (2020). Disease risk analysis in sea turtles: a baseline study to inform conservation efforts. PloS One 15, e0230760. doi: 10.1371/journal.pone.0230760
Mashkour N., Jones K., Wirth W., Burgess G. W., Ariel E. (2021). The concurrent detection of Chelonid alphaherpesvirus 5 and Chelonia mydas papillomavirus 1 in tumoured and non-tumoured green turtles. Anim. 11, 697. doi: 10.3390/ani11030697
Mashkour N., Maclaine A., Burgess G. W., Ariel E. (2018). Discovery of an Australian Chelonia mydas papillomavirus via green turtle primary cell culture and qPCR. J. Virol. Methods 258, 13–23. doi: 10.1016/j.jviromet.2018.04.004
Morrison C. L., Iwanowicz L., Work T. M., Fahsbender E., Breitbart M., Adams C., et al. (2018). Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis. PeerJ 6, e4386. doi: 10.7717/peerj.4386
Ng T. F. F., Manire C. A., Borrowman K., Langer T., Ehrhart L., Breitbart M. (2009). Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics. J. Virol. 83, 2500–2509. doi: 10.1128/JVI.01946-08
Niller H. H., Wolf H., Minarovits J. (2011). Viral hit and run-oncogenesis: genetic and epigenetic scenarios. Cancer Lett. 305, 200–217. doi: 10.1016/j.canlet.2010.08.007
Origlia J. A., Loureiro J. P., Tizzano M. A., Maydup F., Alvarez K., Heredia S. R., et al. (2023). Fibropapillomatosis associated with Chelonid alphaherpesvirus 5 (ChHV5) in a green turtle Chelonia mydas in Argentine waters. J. Wildl. Dis. 59, 363–366. doi: 10.7589/JWD-D-22-00083
Page-Karjian A., Chabot R., Stacy N. I., Morgan A. S., Valverde R. A., Stewart S., et al. (2020a). Comprehensive health assessment of adult female green turtles (Chelonia mydas) nesting in southeastern Florida. Endanger. Species Res. 42, 21–35. doi: 10.3354/esr01036
Page-Karjian A., Gottdenker N. L., Whitfield J., Herbst L. H., Norton T. M., Ritchie B. W. (2017). Potential non cutaneous sites of Chelonid herpesvirus 5 persistence and shedding in green turtles Chelonia mydas. J. Aquat. Anim. Health 29, 136–142. doi: 10.1080/08997659.2017.1321590
Page-Karjian A., Norton T. M., Krimer P., Groner M., Nelson S. E., Gottdenker N. L. (2014). Factors influencing survivorship of rehabilitating green sea turtles (Chelonia mydas) with fibropapillomatosis. J. Zoo Wildl. Med. 45, 507–519. doi: 10.1638/2013-0132R1.1
Page-Karjian A., Norton T. M., Ritchie B. W., Brown C., Mancia C., Jackwood M., et al. (2015). Quantifying Chelonid herpesvirus 5 in symptomatic and asymptomatic rehabilitating green sea turtles. Endanger. Species Res. 28, 135–146. doi: 10.3354/esr00687
Page-Karjian A., Perrault J. R. (2021). “Sea turtle health assessments: Maximizing turtle encounters to better understand health,” in Sea turtle research and conservation. Lessons from working in the field. Nahill B. (Elsevier Academic Press, Cambridge), 31–44.
Page-Karjian A., Perrault J. R., Zirkelbach B., Pescatore J., Riley R., Stadler M., et al. (2019). Tumor re-growth, case outcome, and tumor scoring systems in rehabilitated green turtles with fibropapillomatosis. Dis. Aquat. Org. 137, 101–108. doi: 10.3354/dao03426
Page-Karjian A., Serrano M. E., Cartzendafner J., Morgan A., Ritchie B. W., Gregory C. R., et al. (2020b). Molecular assessment of Chelonid alphaherpesvirus 5 infection in tumor-free green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles in North Carolina, USA 2015–2019. Animals 10, 1964. doi: 10.3390/ani10111964
Patrício A. R., Herbst L. H., Duarte A., Vélez-Zuazo X., Santos Loureiro N., Pereira N., et al. (2012). Global phylogeography and evolution of Chelonid fibropapilloma-associated herpesvirus. J. Gen. Virol. 93, 1035–1045. doi: 10.1099/vir.0.038950-0
Patrício A. R., Velez-Zuazo X., Diez C. E., Dam R. V., Sabat A. M. (2011). Survival probability of immature green turtles in two foraging grounds at Culebra, Puerto Rico. Mar. Ecol. Prog. Ser. 440, 217–227. doi: 10.3354/meps09337
Pérez Y. A. A., Lima S. R., Souza G. M., Gião T., Bispo F. J., Reis A. M. F., et al. (2023). Evaluation of biometry and blood concentration of heavy metals in free-living Chelonia mydas with and without fibropapillomatosis in southern Brazil. Mar. Poll. Bull. 190, 114879. doi: 10.1016/j.marpolbul.2023.114879
Perrault J. R., Levin M., Mott C. R., Bovery C. M., Bresette M. J., Chabot R. M., et al. (2021). Insights on immune function in free-ranging green sea turtles (Chelonia mydas) with and without fibropapillomatosis. Animals 11, 861. doi: 10.3390/ani11030861
Perrault J. R., Stacy N. I., Lehner A. F., Mott C. R., Hirsch S., Gorham J. C., et al. (2017). Potential effects of brevetoxins and toxic elements on various health variables in Kemp’s ridley (Lepidochelys kempii) and green (Chelonia mydas) sea turtles after a red tide bloom event. Sci. Total Environ. 605–606, 967–979. doi: 10.1016/j.scitotenv.2017.06.149
Quackenbush S. L., Casey R. N., Murcek R. J., Paul T. A., Work T. M., Limpus C. J., et al. (2001). Quantitative analysis of herpesvirus sequences from normal tissue and fibropapillomas of marine turtles with real-time PCR. Virol. 287, 105–111. doi: 10.1006/viro.2001.1023
Raiti P. (2008). Carbon dioxide (CO2) laser treatment of cutaneous papillomas in a common snapping turtle, Chelydra serpentina. J. Zoo Wildl. Med. 39, 252–256. doi: 10.1638/2007-0055R.1
Rattner B. A., Heath A. G. (2002). “Environmental factors affecting contaminant toxicity in aquatic and terrestrial vertebrates,” in Handbook of ecotoxicology (Boca Raton: CRC Press), 703–724.
Rees A. F., Alfaro-Shigueto J., Barata P. C. R., Bjorndal K. A., Bolten A. B., Bourjea J., et al. (2016). Are we working towards global research priorities for management and conservation of sea turtles? Endanger. Species Res. 31, 337–382. doi: 10.3354/esr00801
Reséndiz E., Fernández-Sanz H., Domínguez-Contreras J. F., Ramos-Díaz A. H., Mancini A., Zavala-Norzagaray A. A., et al. (2021). Molecular characterization of chelonid alphaherpesvirus 5 in a black turtle (Chelonia mydas) fibropapilloma from Baja California Sur, Mexico. Animals 11, 105. doi: 10.3390/ani11010105
Rivers T. M. (1937). Viruses and koch’s postulates. J. Bacteriol. 33, 1–12. doi: 10.1128/jb.33.1.1-12.1937
Rodenbusch C. R., Baptistotte C., Werneck M. R., Pires T. T., Melo M. T. D., De Ataíde M. W., et al. (2014). Fibropapillomatosis in green turtles Chelonia mydas in Brazil: characteristics of tumors and virus. Dis. Aquat. Org. 111, 207–217. doi: 10.3354/dao02782
Roost T., Schies J.-A., Girondot M., Robin J.-P., Lelong P., Martin J., et al. (2022). Fibropapillomatosis prevalence and distribution in immature green turtles (Chelonia mydas) in Martinique Island (Lesser Antilles). EcoHealth 19, 190–202. doi: 10.1007/s10393-022-01601-y
Rossi S., Sánchez-Sarmiento A. M., Vanstreels R. E. T., dos Santos R. G., Prioste F. E. S., Gattamorta M. A., et al. (2016). Challenges in evaluating the severity of fibropapillomatosis: A proposal for objective index and score system for green sea turtles (Chelonia mydas) in Brazil. PloS One 11, e016763. doi: 10.1371/journal.pone.0167632
Rossi S., Zamana R. R., de Andrade-Santos P. P., da Costa Bomfim A., de Farias D. S. D., da Boaviagem Freire A. C., et al. (2021). Visceral neoplasms and Chelonid alphaherpesvirus 5 in green turtle with fibropapillomatosis. Arch. Vet. Sci. 26, 63–79. doi: 10.5380/avs.v26i1
Sánchez-Sarmiento A. M., Rossi S., Vilca F. Z., Vanstreels R. E. T., Monteiro S. H., Vale L. A. S., et al. (2017). Organochlorine pesticides in green sea turtles (Chelonia mydas) with and without fibropapillomatosis caught at three feeding areas off Brazil. J. Mar. Biolog. Assoc. U. K. 97, 215–223. doi: 10.1017/S002531541500226X
Schneider L., Eggins S., Maher W., Vogt R. C., Krikowa F., Kinsley L., et al. (2015). An evaluation of the use of reptile dermal scutes as a non-invasive method to monitor mercury concentrations in the environment. Chemosphere 119, 163–170. doi: 10.1016/j.chemosphere.2014.05.065
Seminoff J. A. (2023). Chelonia mydas (amended version of 2004 assessment). The IUCN Red List of Threatened Species 2023: e.T4615A247654386. doi: 10.2305/IUCN.UK.2023-1.RLTS.T4615A247654386.en. Available at: www.iucnredlist.org. (Accessed on 22 June 2024).
Serra F., Iaccarino D., Fiorito F., Di Nocera F., Esposito M., Cerracchio C., et al. (2023). Investigation on anthropogenic and opportunistic factors relevant to the incidence of stranded loggerhead sea turtle Caretta caretta along South Tyrrhenian coasts. Front. Mar. Sci. 10. doi: 10.3389/fmars.2023.1116804
Shaver D. J., Walker J. S., Backof T. F. (2019). Fibropapillomatosis prevalence and distribution in green turtles Chelonia mydas in Texas (USA). Dis. Aquat. Org. 136, 175–182. doi: 10.3354/dao03403
Siddell S. G., Lefkowitz E. J., Walker P. J. (2020)Abolish type species. Available online at: https://ictv.global/taxonomy/taxondetails?taxnode_id=202204049.
Smith G. M., Coates C. W. (1938). Fibro-epithelial growths of the skin in large marine turtles, Chelonia mydas (Linnaeus). Zool. 23, 93–98. doi: 10.5962/p.203654
Stacy B. A., Foley A. M., Work T. M., Lauritsen A. M., Schroeder B. A., Hargrove S. K., et al. (2019). Report of the technical expert workshop: Developing recommendations for field response, captive management, and rehabilitation of sea turtles with fibropapillomatosis. US Department Commerce Natl. Mar. Fisheries Service (NOAA Tech. Memorandum NMFS OPR-60), 1–56. doi: 10.25923/adg2-g771
Stammnitz M. R., Coorens T. H. H., Gori K. C., Hayes D., Fu B., Wang J., et al. (2018). The origins and vulnerabilities of two transmissible cancers in Tasmanian devils. Cancer Cell 33, 607–619. doi: 10.1016/j.ccell.2018.03.013
Turakhia Y., Thornlow B., Hinrichs A., McBroome J., Ayala N., Ye C., et al. (2022). Pandemic-scale phylogenomics reveals the SARS-CoV-2 recombination landscape. Nature 609, 994–997. doi: 10.1038/s41586-022-05189-9
Van Houtan K. S., Smith C. M., Dailer M. L., Kawachi M. (2014). Eutrophication and the dietary promotion of sea turtle tumors. PeerJ 2, e602. doi: 10.7717/peerj.602
Vanstreels R. E. T., Durant A., Santos A. P., Santos R. G., Sarmiento A. M. S., Rossi S., et al. (2023). Exploring the relationship between environmental drivers and the manifestation of fibropapillomatosis in green turtles (Chelonia mydas) in eastern Brazil. PloS One 18, e0290312. doi: 10.1371/journal.pone.0290312
Whitmore L., Yetsko K., Farrell J. A., Page-Karjian A., Daniel W., Shaver D. J., et al. (2021). Evolutionary comparisons of Chelonid alphaherpesvirus 5 (ChHV5) genomes from fibropapillomatosis-afflicted green (Chelonia mydas), olive ridley (Lepidochelys olivacea) and Kemp’s ridley (Lepidochelys kempii) sea turtles. Animals 11, 2489. doi: 10.3390/ani11092489
Work T. M., Ackermann M., Casey J., Chaloupka M., Herbst L., Lynch J., et al. (2015a). The story of invasive algae, arginine, and turtle tumors does not make sense. PeerJ PrePrints 2, e539v1. doi: 10.7287/peerj.preprints.539v1
Work T. M., Balazs G. H., Rameyer R. A., Morris R. A. (2004). Retrospective pathology survey of green turtles Chelonia mydas with fibropapillomatosis in the Hawaiian Islands 1993-2003. Dis. Aquat. Org. 62, 163–167. doi: 10.3354/dao062163
Work T. M., Balazs G. H., Wolcott M., Morris R. (2003). Bacteraemia in free-ranging Hawaiian green turtles Chelonia mydas with fibropapillomatosis. Dis. Aquat. Org. 53, 41–46. doi: 10.3354/dao053041
Work T. M., Dagenais J., Balazs G. H., Schettle N., Ackermann M. (2014). Dynamics of virus shedding and in-situ confirmation of chelonid herpesvirus 5 in Hawaiian green turtles with fibropapillomatosis. Vet. Pathol. 52, 1195–1220. doi: 10.1177/0300985814560236
Work T. M., Dagenais J., Balazs G. H., Schettle N., Ackermann M. (2015b). Dynamics of virus shedding and in situ confirmation of Chelonid herpesvirus 5 in Hawaiian green turtles with fibropapillomatosis. Vet. Pathol. 52, 1195–1201. doi: 10.1177/0300985814560236
Work T. M., Dagenais J., Balazs G. H., Schumacher J., Lewis T. D., Leong J.-A. C., et al. (2009). In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas). J. Gen. Virol. 90, 1943. doi: 10.1099/vir.0.011650-0
Work T. M., Dagenais J., Weatherby T. M., Balazs G. H., Ackermann M. (2017). In vitro replication of Chelonid herpesvirus 5 in organotypic skin cultures from Hawaiian green turtles (Chelonia mydas). J. Virol. 91, 10–1128. doi: 10.1128/JVI.00404-17
Work T. M., Dagenais J., Willimann A., Balazs G. H., Mansfield K., Ackermann M. (2020). Differences in antibody responses against Chelonid alphaherpesvirus 5 (ChHV5) suggest differences in virus biology in ChHV5-seropositive green turtles from Hawaii and ChHV5-seropositive green turtles from Florida. J. Virol. 94, e01658–e01619. doi: 10.1128/JVI.01658-19
Work T. M., Rameyer R. A., Balazs G. H., Cray C., Chang S. P. (2001). Immune status of free ranging green turtles with fibropapillomatosis from Hawaii. J. Wildl. Dis. 37, 574–581. doi: 10.7589/0090-3558-37.3.574
Yan M., Nie H., Wang W., Huang Y., Li Q. X., Wang J. (2018). The risk of polychlorinated biphenyls facilitating tumors in Hawaiian green sea turtles (Chelonia mydas). Int. J. Environ. Res. Public Health 15, 1243. doi: 10.3390/ijerph15061243
Yetsko K., Farrell J. A., Blackburn N. B., Whitmore L., Stammnitz M. R., Whilde J., et al. (2021). Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors. Commun. Biol. 4, 152. doi: 10.1038/s42003-021-0
Keywords: sea turtle, conservation, fibropapillomatosis, viral agent, pollution, veterinary medicine, host genomics
Citation: Dupont SM, Bustamante P, Duffy DJ, Fort J, Le Loc’h G, Lelong P, Chevallier D and Giraudeau M (2024) Future research avenues for the study of fibropapillomatosis in sea turtles. Front. Ecol. Evol. 12:1372096. doi: 10.3389/fevo.2024.1372096
Received: 17 January 2024; Accepted: 12 June 2024;
Published: 10 July 2024.
Edited by:
Hector Barrios-Garrido, University of Zulia, VenezuelaReviewed by:
Vanessa Labrada Martagón, Autonomous University of San Luis Potosí, MexicoMaximilian Polyak, General Oragnization for the Conservation of Corals and Sea Turtles in the Red Sea, Saudi Arabia
Copyright © 2024 Dupont, Bustamante, Duffy, Fort, Le Loc’h, Lelong, Chevallier and Giraudeau. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Sophie M. Dupont, c29waGllLmR1cG9udDkzQGdtYWlsLmNvbQ==
†These authors have contributed equally to this work
‡These authors are listed alphabetically