Rachel Thomas's research while affiliated with University of Florida and other places

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Publications (16)


Detection and population genomics of sea turtle species via non‐invasive environmental DNA analysis of nesting beach sand tracks and oceanic water
  • Article

April 2022

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326 Reads

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26 Citations

Molecular Ecology Resources

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

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Supplemental Figure 1.jpg
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October 2021

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3 Reads

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Fig. 1. Green sea turtles undergoing rehabilitation at the Whitney Laboratory Sea Turtle Hospital. Top: In the absence of enrichment, turtles have minimal environmental stimulation. Bottom: Green turtle interacting with food ice block.
Fig. 2. Behavioural responses to food-based enrichment. A) Average time (seconds) spent by all turtles exhibiting each behaviour with no enrichment or frozen food enrichments (five turtles). Behaviours have been categorised as 'positive' or 'negative'. (Five turtles; per turtle observation N = 5; each observation = 20 min). All statistically significant changes have been indicated with the p-value above (Wilcoxon signed-rank test). B) Activity budget of five green turtles (C. mydas) when observed without enrichment or with food ice blocks.
Fig. 3. Patient 6 case study. A) Average time (seconds) spent by Patient 6 exhibiting each behaviour across five conditions (no enrichment, frozen food [ice greens], sea lettuce gate, floating whiffle ball with food and sunken whiffle ball with food). Behaviours have been categorised as 'positive' or 'negative'. N = 5 for no enrichment and sunken whiffle ball, N = 1 for all other enrichments; each observation = 20 min). All statistically significant (and near significant) changes have been indicated with the p-value above the comparisons (paired t-test). B) Image of Patient 6 feeding from sunken whiffle ball enrichment.
Effects of food-based enrichment on the behaviour of juvenile green turtles (Chelonia mydas) in a rehabilitation facility in Florida

October 2021

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450 Reads

Wild sea turtles that are admitted to turtle hospitals and rehabilitation centers suffering from illnesses and injuries may be held for extended periods of months to years, until they are recovered and ready for release back to the wild. During this time, natural behaviors may be limited, potentially adversely affecting the long-term rehabilitation success, however, little research has been carried out on the behavior of hospitalized sea turtles. Here we report that environmental enrichment can be an effective means of encouraging natural behaviors in turtles in hospital/rehabilitation and aquarium settings, but find that enrichment should be monitored and tailored to individual turtles to achieve positive results.






Fig. 2 Environmental DNA (eDNA)-based detection, quantification, and monitoring of ChHV5 viral shedding into patient tank water, and sand. a Correlation of individual patient tumor surface area (mm 2 ) and the concentration of ChHV5 virus being shed into their tank water, as detected by ChHV5 UL30 gene DNA qPCR (positive correlation, Pearson correlation coefficient test, R 2 = 0.5431, p = 0.0002, df = 19). Water containing virus schematic insert was generated using BioRender (https://biorender.com/). b Detection of ChHV5 viral shedding onto sand which a patient ("Archie Carr" 49-2020-Cm) temporarily lay on while awaiting treatment. Detection of ChHV5 UL30 gene DNA by qPCR. Amplification ratios for each sand sample are provided in Supplementary Table 1. Error bars denote the standard deviation of six technical replicates. c Patient time-course of tumor surface area changes (surgical removal) and concentration of ChHV5 shed into tank water, as detected by UL30 qPCR. d Time-course of ChHV5 viral shedding into four patient tanks, as detected by UL30 qPCR. Tumor removal surgery events are denoted by an asterisk. Note: the ChHV5 eDNA detected in tank 1 in week 4 was due to a second patient (FP-positive) being added to that tank for a single week, due to the rehabilitation needs of the hospital. Error bars denote the standard deviation of three biological samplings, each with three qPCR technical replicates. e Prolonged patient time-course of tumor surface area changes (surgical removal) and concentration of ChHV5 shed into tank water, as detected by UL30 qPCR. f Correlation of Chelonia mydas (green sea turtle) eDNA shedding and ChHV5 eDNA shedding (positive correlation, Pearson correlation coefficient test, R 2 = 0.66, p = 0.00001, df = 19), both C. mydas (16S rRNA gene assay) and ChHV5 (UL30 gene assay) eDNA were detected by qPCR.
Fig. 5 Variation in ChHV5 DNA load across tumor and non-tumor tissue types, as assessed by whole-genome sequencing and qPCR. a ChHV5 abundance (WGS DNA-based) across each sample type. Graph of the number of ChHV5 reads (DNA-seq) per 10 million total reads of 35 samples, with Tukey whiskers. Per sample type: external fibropapillomatosis tumor n = 7, internal fibropapillomatosis tumor n = 6 (4 kidney FP, 1 bladder FP and 1 lung FP), non-tumor n = 8 (2 skin, 2 kidney, 1 bladder, 1 lung, and 2 whole blood), plasma from FP-afflicted turtles n = 10, tissue from non-tumor hatchlings n = 3 (1 green, 1 loggerhead, and 1 leatherback), tank environmental DNA (eDNA) n = 1 (pooled sample of eDNA from 4 tanks with FP-afflicted juvenile green patients and 1 tank with FP-free loggerhead hatchlings). P-values for all groups with significant differences (t-test) to the non-tumor (skin and whole blood) sample cohort are shown on the graph. All samples are from green sea turtles, with the exception of two of the hatchling samples (loggerhead and leatherback), and the eDNA tank sample being pooled from four green and one loggerhead tank water extractions (Supplementary Fig. 2c). b Image of new growth tumors on patient "Lilac" (25-2018-Cm). "Lilac" was admitted to the hospital without tumors, but with a number of leech bites which later developed into FP tumors. ChHV5 relative quantification of "Lilac" new growth tumor samples and non-tumor skin punch biopsy samples using the UL30 DNA qPCR assay 37 (Supplementary Table 6). Error bars denote the standard deviation of three qPCR technical replicates. ChHV5 DNA was detected in these growths via qPCR prior to them being classified as fibropapillomatosis by histology. c ChHV5 viral quantification box plot with Tukey whiskers of a range of FP types (n = 43 samples) and non-tumor tissue (n = 36) samples from 13 individual patients using the UL30 DNA qPCR assay 37 (Supplementary Table 6). Individual sample values within each sample type are shown by the open points. Absolute quantity of ChHV5 was determined through a standard curve of known amounts (in picograms) of a UL30 gene fragment (Supplementary Table 6). Truncated x-axis label, internal sub-cutane. = internal subcutaneous.
Leech ChHV5 detection
a Green sea turtle inguinal external FP tumor, infested with leeches. Upon patient intake FP-afflicted tumors frequently harbor marine leeches, as was the case for this patient 02-2021-Cm “Broccoli”. Leeches from “Broccoli’s” tumors were used for the ChHV5 analysis in (e). Leeches are commonly found within the crevasses of external FP tumors (right image). b Detailed view of a marine leech removed from the surface of a fibropapillomatosis tumor, with gills and with dark red blood pellet (after feeding on a C. mydas turtle) visible. c Detection and quantification of ChHV5 UL30 gene DNA by qPCR, using leeches as proxy eDNA samples (whole leech lysis and DNA extraction). Error bars denote the standard deviation of three technical replicates. Amplification ratio for leeches from the FP-free loggerhead turtle (09-2015-Cc) was 0, and the amplification ratio for both the FP-tumor leech and FP-tumor tissue samples (green turtle, 07-2015-Cm) was 1.0. d Quantification of ChHV5 UL30 gene DNA by qPCR, from leeches removed from FP-afflicted green turtles from either FP tumors or non-tumor locations. Individual turtle denoted by rd—36-2020-Cm “Richard Dawkins”, bh—52-2020-Cm “Bruno Hofer”, or rg—78-2020-Cm “Ruth Gates”. Approximately ten leeches were pooled for each of the 12 DNA extraction samples. Error bars denote the standard deviation of six technical replicates. e Quantification of ChHV5 UL30 gene DNA by qPCR, from leeches (individual leech DNA extractions) removed from FP tumors of green sea turtle patient 02-2021-Cm “Broccoli” (a). Amplification ratios for leech samples are provided in Supplementary Table 1. Error bars denote the standard deviation of six technical replicates.
Transcriptomics of ChHV5 in external and internal FP tumors, and non-tumor tissue samples
a ChHV5 expression across each sample type. Box plot with Tukey whiskers of the number of ChHV5 reads (RNA-seq) per 10 million total reads per sample. Individual sample values within each sample type are shown by the open points. Significant differences in averages between sample types were determined by a Kruskal–Wallis with Dunn–Bonferroni post hoc test and are denoted by an asterisk (*). Per sample type: non-tumor n = 20, established external tumor n = 35, regrowth external tumor n = 12, new growth external tumor n = 9, kidney tumor n = 6, lung tumor n = 7. b Top: Rehabilitating FP-afflicted juvenile green sea turtle. Image credit: Nancy Condron. Bottom: Hematoxylin and eosin stained fibropapillomatosis tumor, external regrowth tumor. c Total number of samples in which reads (RNA-Seq) for each ChHV5 gene were detected. A gene was counted as detected if a sample had TPM-normalized counts >0 for said ChHV5 gene.
Patient rehabilitation outcome and ChHV5 gene expression
a Box plot with Tukey whiskers of the number of ChHV5 reads (RNA-Seq) per 10 million total reads between patients based on outcome (released vs died in care/humanely euthanized). Individual sample values within each sample type are shown by the open points. Significant difference in averages between the two outcomes was determined by a Mann–Whitney U test and is denoted by an asterisk (*). N = 69 samples. Per outcome: released = 7 turtles; died/euthanized = 5 turtles. b Box plot with Tukey whiskers of the expression levels of the ChHV5 F-LANA genes (both copies of the F-LANA gene: F-LANA and F-LANA’) in transcripts per million (TPM), by patient outcome and tumor location, as detected by RNA-seq. Individual sample values within each sample type are shown by the open points. c Principal component analysis (PCA) of the viral transcriptomes (minus 28 genes which had 0 reads across all samples, see methods) of all tumor samples (RNA-seq), by tumor location and patient outcome.
Environmental DNA monitoring of oncogenic viral shedding and genomic profiling of sea turtle fibropapillomatosis reveals unusual viral dynamics

May 2021

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327 Reads

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24 Citations

Communications Biology

Pathogen-induced cancers account for 15% of human tumors and are a growing concern for endangered wildlife. Fibropapillomatosis is an expanding virally and environmentally co-induced sea turtle tumor epizootic. Chelonid herpesvirus 5 (ChHV5) is implicated as a causative virus, but its transmission method and specific role in oncogenesis and progression is unclear. We applied environmental (e)DNA-based viral monitoring to assess viral shedding as a direct means of transmission, and the relationship between tumor burden, surgical resection and ChHV5 shedding. To elucidate the abundance and transcriptional status of ChHV5 across early, established, regrowth and internal tumors we conducted genomics and transcriptomics. We determined that ChHV5 is shed into the water column, representing a likely transmission route, and revealed novel temporal shedding dynamics and tumor burden correlations. ChHV5 was more abundant in the water column than in marine leeches. We also revealed that ChHV5 is latent in fibropapillomatosis, including early stage, regrowth and internal tumors; higher viral transcription is not indicative of poor patient outcome, and high ChHV5 loads predominantly arise from latent virus. These results expand 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 species.


Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors

February 2021

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230 Reads

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34 Citations

Communications Biology

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression. Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFβ and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers. Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.


Analysis of plastic pieces identified from post-hatchling washback necropsies. Post-hatchling washback sea turtles. (A) Post-hatchling washback Caretta caretta sea turtles being released back into the Atlantic Ocean, North Florida, after being successfully rehabilitated at the University of Florida’s Whitney Laboratory Sea Turtle Hospital. (B) A deceased loggerhead (C. caretta) washback with scale bar, prior to necropsy. Dashed white line indicates maximum straight carapace length. (C) Intact intestinal tract of a loggerhead washback with arrows indicating presence of plastic. (D) Extracted gastrointestinal tract from a loggerhead necropsy, with scale ruler for size comparison, each box on the scale ruler is 1 cm². (E) Comparison of the quantity of plastic pieces ingested by washbacks in 2016, compared to 2017. (F) Comparison of the dry mass of ingested plastic pieces in washbacks in 2016, compared to 2017. (G) Comparison of the proportion of ingested plastic to washback weight in 2016 washbacks, compared with 2017 washbacks. Boxplots were generated using BoxPlotR (Spitzer et al., 2014).
Analysis of plastic types and hard plastic size ingested by post-hatchling washbacks. (A) Quantity of each type of plastic ingested by washbacks (2016 and 2017). (B) Quantity of washbacks that ingested each type of plastic type (2016 and 2017). (C) Percentage of macroplastic versus microplastic ingested (2016 and 2017). (D) Comparison of the maximum size of hard plastic ingested by 2016 washbacks, compared to 2017 washbacks. (E) Correlation of ingested hard plastic size to corresponding washback size (2016 and 2017).
Analysis of plastic ingestion recovery location and color in post-hatchling washbacks. (A) Quantity of plastic pieces of each color recovered from washback necropsies (2016 and 2017). (B) Quantity of plastic pieces recovered from each gastrointestinal tract location (2017).
Comparison of sea turtle plastic ingestion incidence across research studies.
Plastic Ingestion in Post-hatchling Sea Turtles: Assessing a Major Threat in Florida Near Shore Waters

August 2020

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466 Reads

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43 Citations

Frontiers in Marine Science

Frontiers in Marine Science

Pollution from anthropogenic marine debris, particularly buoyant plastics, is ubiquitous across marine ecosystems. Due to the persistent nature of plastics in the environment, their buoyancy characteristics, degradation dynamics, and ability to mimic the behavior of natural prey, there exists significant opportunity for marine organisms to ingest these man-made materials. In this study we examined gastrointestinal (GI) tracts of 42 post-hatchling loggerhead (Caretta caretta) sea turtles stranded in Northeast Florida. Necropsies revealed abundant numbers of plastic fragments ranging from 0.36 to 12.39 mm in size (length), recovered from the GI tracts of 39 of the 42 animals (92.86%), with GI burdens ranging from 0 to 287 fragments with a mass of up to 0.33 g per turtle. Post-hatchlings weighed from 16.0 to 47.59 g yielding a plastic to body weight percentage of up to 1.23%. Several types of plastic fragments were isolated, but hard fragments and sheet plastic were the most common type, while the dominant frequency of fragment color was white. Fragment size and abundance mixed with natural gut contents suggests significant negative health consequences from ingestion in animals at this life stage. Gaining greater insight into the prevalence of plastic ingestion, the types of plastic and the physiological effects of plastic consumption by multiple life-stages of sea turtles will aid the prioritization of mitigation efforts for the growing marine debris problem. This report demonstrates that plastic ingestion is a critical issue for marine turtles from the earliest stages of life.


Citations (8)


... There are currently two hypotheses as follows: horizontal transmission through direct contact with tumors/secretions or through vectors and water [53], and vertical transmission as recent analyses have reported the presence of ChHV5 in hatchlings [60]. However, in another study, the hatchlings tested negative despite coming from mothers that tested positive using the same method [61]. ...

Reference:

Fibropapillomatosis: A Review of the Disease with Attention to the Situation Northern Coast of Brazil
Detection and population genomics of sea turtle species via non‐invasive environmental DNA analysis of nesting beach sand tracks and oceanic water
  • Citing Article
  • April 2022

Molecular Ecology Resources

... To date, at least one virus, chelonid alphaherpesvirus 5 (ChHV5), has been strongly associated with FP while other viruses such as Chelonia mydas papillomavirus 1 (CmPV1), retrovirus and sea turtle tornovirus 1 have been found in tumor tissues of green turtles with no clear association to disease development, pattern, and severity [30][31][32][33][34][35][36][37][38][39][40] . Disease manifestation is likely to be multifactorial, as the presence of ChHV5 alone is not linked with tumor formation, and recovery and regression are possible in some cases 25,[41][42][43][44][45] . Anthropogenic stressors, environmental pollutants, immunosuppression and genetic predisposition are suggested as co-factors in tumor growth and disease prevalence 15, 26 . ...

Environmental DNA monitoring of oncogenic viral shedding and genomic profiling of sea turtle fibropapillomatosis reveals unusual viral dynamics

Communications Biology

... Considering the historical data, it is likely that ChHV5/ Scutavirus chelonidalpha5 has been passively spreading (possibly in latency or lethargy stage) for decades in the feeding sites of NW Mexico, with or without the sporadic manifestation of FP (Alfaro-Núñez et al. 2016) but not efforts were consistently made to detect the presence of the viral disease. In this sense, infected turtles in latently stage that later on began to develop visible tumours, could be related to unknown environmental and/or host immunity factors (Alfaro-Núñez et al. 2016;Lawrance et al. 2018;Yetsko et al. 2020Yetsko et al. , 2021. Non-infectious factors appear to play an important role in the pathogenesis of virus-associated neoplasia diseases (such as FP) and are often related to pollution caused by human activities, which may generate a change (or imbalance) in host-pathogen interactions via immunosuppression, resulting in a potential increased disease frequency and/or severity with serious consequences for the animal, environmental, and human health (Bossart 2011;Bossart and Duignan 2019;Hamede et a. 2020;Baines et al. 2021). ...

Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors

Communications Biology

... For example, Coc et al. (2021) described microplastics from a marine detritivore. Even some herbivorous organisms have been observed to have microplastics present in their systems, including manatees (Bergmann et al. 2015) and sea turtles (Caron et al. 2018;Eastman et al. 2020). There is an increased need for research and 92 caribbean Journal of science [Volume 54 ...

Plastic Ingestion in Post-hatchling Sea Turtles: Assessing a Major Threat in Florida Near Shore Waters
Frontiers in Marine Science

Frontiers in Marine Science

... A PHA skin test stimulates T-cell proliferation, differentiation, and cytokine production with inflammation and swelling at the injection site. The accumulation of lead (Pb) in kidneys may be immunosuppressive and disrupt T-lymphocyte proliferation [212]. ...

Mutational, transcriptional and viral shedding dynamics of the marine turtle fibropapillomatosis tumor epizootic

... Viral diseases in sea turtles include fibropapillomatosis (FP), which consists of multifocal cutaneous or visceral tumors in juvenile or adult animals. This neoplasia is benign, but depending on its size, number and location, it can cause problems such as impaired vision, diving and feeding [20,[35][36][37]. The FP is distributed worldwide among sea turtles. ...

Behaviour of juvenile green turtles (Chelonia mydas) before and after fibropapillomatosis tumour removal

... From the data collected in our study, a maximum of 15 tumours per individual were identified, this is low compared to cases reported in Brazil, where up to 129 tumours per turtle have been found (Rossi et al. 2016), but it coincides with reports from Costa Rica (Brenes et al. 2013), where a maximum of 12 tumours per individual was found in nesting olive ridley turtles. The predominance of small tumours is consistent with the findings in other studies from the Mexican Pacific (Gámez et al. 2009) and Gulf of Mexico (Suárez-Domínguez et al. 2020) but differ from those reported in places where the prevalence of FP is high, such as Hawaii and Florida, where it is common that tumours exceed 10 cm in diameter (Work et al. 2004;Farrell et al. 2018). The differences between the sizes of the tumours may be because the turtles in regions like Hawaii and Florida have presence of the disease for a longer time, and as the disease progresses, the lesions increase in size (Farrell et al. 2018). ...

Characterisation of fibropapillomatosis tumour growth profiles in green sea turtles (Chelonia mydas)

... This is due to the ability of radiation to damage DNA and thus give rise to an increase in the mutations that cause tumors in animals. In addition, in captive turtles, tumor growth was more evident during warmer times of the year, indicating that climate change can directly impact FP [67][68][69]. ...

Sea turtle fibropapilloma tumors share genomic drivers and therapeutic vulnerabilities with human cancers

Communications Biology