What if Jurassic Park wasn’t so much science-fiction as it was science-fact? Bringing long extinct animals back to life is simply fiction (for a little longer at least, given the rate of progress of de-extinction efforts¹). But, did you know that molecular science has already evolved and adapted to be able to detect animals that have disappeared, thought to be extinct for over half a century, but in fact are still alive unbeknownst to us²? Even more unbelievable is that other fictional/mythological creatures – like the Loch Ness Monster – may truly be out there, and discoverable by these scientific methodologies already at play³. This bridging of fantasy and science is partly possible thanks to the development and optimization of a relatively novel genetic technology involving environmental (e)DNA⁴. Every organism sheds their DNA into their environment, whether they’re a human, a not-so-extinct frog or even a mythological lake monster⁴. Skin, hair, scales and bodily fluids/excretions are shed into every environment – from ocean water to arctic snow to desert sand – and are extractable, detectable and readable by the unique application of relatively standard molecular and genetic techniques⁴. So, while there are a few big-mystery investigations that can begin with the application of eDNA approaches, there are many more “down to earth” questions that can be addressed in the less fantastical scientific world – from detecting and tracking elusive, endangered species, to learning more about habitat ranges of various populations, to monitoring pathogens and diseases transmitted throughout the environment^4,5.

Here at the University of Florida’s (UF) Whitney Laboratory for Marine Bioscience and Sea Turtle Hospital⁶, we wondered if we could adapt the process to help the vulnerable species we treat every day – green sea turtles (Chelonia mydas). While all sea turtle species face a substantial array of threats – from plastic pollution⁷ to predation or boat strikes – one threat of particular significance to green sea turtles around the world is a debilitating tumor-causing disease known as fibropapillomatosis (FP)^8-11. Before joining the research team at UF’s Whitney Lab, I had never heard of such a thing and whenever I thought of sea turtles I thought of care-free reptiles who have survived millions of years happily swimming in warm ocean waters. So, when I met my first patient at the Whitney Sea Turtle Hospital, I was astounded by the severity of the tumor burden this disease can cause. While the burden of the disease varies between sea turtle species and individual turtles, since the 1930s it has spiraled to the point that it now impacts all seven sea turtle species in every ocean around the world (panzootic). The disease continues to spread to further geographic locations in which it has never before been recorded. It is now also not only infecting the typical life-stage with more severity (juvenile green turtles) but is infecting more individuals at older stages as well as all other species. Unfortunately, although the disease has been documented and monitored for almost a century, little progress has been made in terms of mitigating its transmission as sea turtles themselves are highly mobile, hard to access aquatic animals, and the pathogen thought to be responsible for triggering the tumor formation remains somewhat of a mystery. A sea turtle specific alphaherpervirus (Chelonid herpesvirus 5, ChHV5), which co-evolved with the sea turtle clade, is currently the most likely etiological agent of FP, and when combined with immunological stress (potentially from anthropogenic factors) triggers lesion formation. However, due to the difficulty in culturing ChHV5 in a lab, little is known about the transmission of this virus. Thinking outside of the usual avenues of viral and immunological investigation, we decided to see if an eDNA approach could give us any insight into viral transmission, with any answers helping to elucidate FP mitigation strategies applicable to our sea turtle patients, and potentially wild populations⁵.

The first part of the project required adapting and optimizing pre-existing eDNA methodologies^12-14 to best suit our target organism – in this case the ChHV5 virus from C. mydas – and our environment – marine tank water. Starting from a protocol for freshwater eDNA sampling, generously provided by Mathew Seymour^15,16, we spent several months optimizing the collection and filtration methodology for our sea water tanks and conducting too many 384-well qPCRs to count. However, once we were confident with our methods, we set out to answer a long list of questions. Could we even detect ChHV5 in our tank water? If so, could we quantify the levels being shed? Was there any relationship between the concentration of ChHV5 present in a patient’s tank and their disease severity/tumor burden? Many of our patients undergo surgery to remove the tumors so that we can release them back into the wild – does this have any effect on the level of viral shedding into their tank environment? And to our pleasant surprise – we were able to definitively answer each of these questions^5,17. We determined that ChHV5 is shed into the water column by our green sea turtle patients, suggesting that direct viral transmission is likely. Not only could we detect the virus being shed into the water, but we were able to quantify and monitor its fluctuations over time as the patients’ disease severity altered as a result of natural processes or surgery. The level of viral eDNA being shed into the tank water was positively correlated to the disease severity of our turtles – in this case this meant that the greater the tumor surface area on the patient, the more ChHV5 they were shedding into the water, and when tumors were surgically removed, the levels of environmental virus drastically decreased. We saw these patterns in every patient that we sampled, whether they were a relatively short-term patient or a turtle who had an extended stay (well over one year), like hospital-favorite patient “Edward Scissorhands” who provided us with our longest-duration sample series as well as lots of heart-warming interactions during her time at the Whitney Lab Sea Turtle Hospital.

Combining the eDNA results with more traditional tumor tissue-based genomic and transcriptomic investigation allowed us to better understand the role of the ChHV5 virus both within the patients themselves and in the environment^5,9. We could link specific host and viral gene expression with patient outcomes (successful versus unsuccessful rehabilitation), correlate immune functioning and viral load and reveal the sea turtle genes responsible for maintaining tumor growth^5,9. However, like many scientific endeavors, the results raised as many new questions as they answered, such as why turtles with lower levels of ChHV5 gene expression have worse outcomes (we suspect viral immune evasion strategies), and if ChHV5 is predominantly latent in FP, why do we detect so much virus in tank water (possibly due to the proportion of free virus compared with that contained within shed sea turtle cells)?

The results from this project have expanded our knowledge of the cellular and shedding dynamics of ChHV5 and can provide insights into temporal transmission dynamics and viral oncogenesis not readily investigable in tumors of terrestrial species⁵. However, this is just the beginning of eDNA applications for the Whitney Laboratory and the field of sea turtle conservation in general. We, and others, are expanding the use of eDNA approaches for the study of wildlife and their pathogens⁴.

While we were able to make significant advances with our eDNA approaches – our progress was steady by wildlife conservation standards – this is perhaps not nearly as impressive as what has occurred in the human pathogen field in the last year. We were intrigued to see that, as a result of the Covid-19 pandemic, a similar application of the eDNA approach we employed – but in this case eRNA – had begun to be used to detect, track and monitor the spread of the Covid-19 pathogen (SARS-CoV-2) through human wastewater systems^17-20. Coronavirus eRNA in wastewater samples could be detected at least a month in advance of the first diagnoses of the disease in several locations¹⁸. This novel application of eRNA has been able to provide early warning and predictive monitoring to the human medical field in a time when humans need it most, and it has been fascinating to watch a technique that was originally developed for wildlife conservation^4,12, and adapted by our team to tackle a sea turtle panzootic, be advanced to play a crucial role in the mitigation of the Covid-19 human pandemic.

It is evident that although eDNA methodologies began developing in the 1980s, their potential continues to grow as new technologies become available, more accessible and affordable. Environmental DNA approaches are being applied to ever more questions from vastly different fields of research – from marine reptile conservation to human healthcare. We look forward to learning of more innovative uses of eDNA for ecological, evolutionary and health related questions, and will continue to combine eDNA with more conventional tissue-based genomics to further elucidate the mysteries of sea turtle fibropapillomatosis. Pathogen eDNA highlights how basic science can have surprising and unexpected applications, and how whether you are animal or human we share a vulnerability to both pathogens and the ill health of the planet.

References

1. https://reviverestore.org/
2. https://onlinelibrary.wiley.com/doi/10.1111/mec.15594
3. https://www.nationalgeographic.com/science/article/loch-ness-monster-scotland-environmental-dna-science
4. https://academic.oup.com/bioscience/advance-article/doi/10.1093/biosci/biab027/6236036
5. https://doi.org/10.1038/s42003-021-02085-2
6. https://www.whitney.ufl.edu/conservation--sea-turtle-hospital/
7. https://www.frontiersin.org/articles/10.3389/fmars.2020.00693/full
8. https://www.academia.edu/41560065/Characterisation_of_fibropapillomatosis_tumour_growth_profiles_in_green_sea_turtles_Chelonia_mydas
9. https://www.nature.com/articles/s42003-021-01656-7

10. https://www.nature.com/articles/s42003-018-0059-x
11. https://www.sciencedirect.com/science/article/pii/S1090023315004529?via%3Dihub
12. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041781
13. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041732
14. https://www.mdpi.com/1999-4915/11/6/526
15. https://www.nature.com/articles/s42003-017-0005-3

16. https://www.slu.se/en/ew-cv/mathew-seymour2/
17. https://theconversation.com/environmental-dna-how-a-tool-used-to-detect-endangered-wildlife-ended-up-helping-fight-the-covid-19-pandemic-158286
18. https://pubmed.ncbi.nlm.nih.gov/32387778/
19. https://www.medrxiv.org/content/10.1101/2020.07.10.20150573v2
20. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229723/