Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

COMPARISON OF AGAROSE GEL AND CAPILLARY ZONE ELECTROPHORESIS METHODS USING PLASMA FROM GREEN TURTLES (CHELONIA MYDAS).

Agarose gel electrophoresis (AGE) has been widely implemented throughout veterinary medicine and for analysis of plasma proteins of avian and reptile species. Capillary zone electrophoresis (CZE) is becoming a standard method in human clinical pathology laboratories but has not widely been used for the analysis of animal samples. The objective of the present study was to compare protein fractions derived from AGE and CZE methods using plasma from the green turtle (Chelonia mydas). Plasma samples were analyzed by AGE and CZE per manufacturer guidelines. The methods were assessed by CV analysis, Spearman's correlation, Passing-Bablok regression, and Bland Altman plots. CZE consistently resolved more fractions than AGE with three fractions observed in the prealbumin migrating region versus one for AGE and two fractions in the γ globulin region versus one for AGE. Compared with AGE, CZE showed a lower CV in intra-assay tests (1.0-4.9% vs 2.0-28.3%) and a lower or overlapping CV in interassay tests (1.0-10.6 vs 2.3-22.0). The prealbumin, α2 globulin, and ß globulin fractions correlated the least between the methods (for all three fractions: rs ≤ 0.28, P > 0.21). Moderate, significant correlations between AGE and CZE methods were observed for albumin (rs = 0.78, P < 0.0001) and γ globulins (rs = 0.78, P < 0.0001). CZE has a higher precision and ease of use over AGE and offers the opportunity to resolve additional protein fractions. This will necessitate the development of new conventions in placement of fraction delimits, definition of species-specific reference intervals, and evaluation of clinical utility in abnormal turtles.

Tumor re-growth, case outcome, and tumor scoring systems in rehabilitated green turtles with fibropapillomatosis.

Fibropapillomatosis (FP) is an infectious, neoplastic disease of major concern in sea turtle rehabilitation facilities. Rehabilitating sea turtles that undergo tumor removal surgery often have tumor regrowth and may experience mortality. We evaluated tumor score, removal, and regrowth in rehabilitating green sea turtles with FP in 4 rehabilitation facilities in the southeastern USA during 2009-2017. Of 756 cases, 312 (41%) underwent tumor removal surgery, 155 (50%) of those had tumor regrowth within an average of 46 ± 45 d, and 85 (27%) had multiple (>1) regrowth events. Of 756 turtles with FP, 563 (75%) did not survive after admission into a rehabilitation facility, including 283 (37%) that were euthanized and 280 that died without euthanasia (37%), and 193 survived, including 186 (25%) released and 7 (1%) placed in permanent captive care. Tumor removal surgery increased the odds of tumor regrowth but also enhanced survivorship, whereas tumor regrowth was not a significant predictor of case outcome. Three FP tumor scoring systems were used to assign tumor scores to 449 cases, and differing results emphasize that tumor scoring systems should be applied to the situations and/or location(s) for which they were intended. FP tumor score was not a significant predictor for the event or extent of FP tumor regrowth after surgical excision. Under current rehabilitation regimes, outcomes of rehabilitation for tumored turtles have a low probability of success. The results of this study may be used to help guide clinical decision-making and determine prognoses for rehabilitating sea turtles with FP.

PLASMA COLLOID OSMOTIC PRESSURE IN CHRONICALLY DEBILITATED LOGGERHEAD SEA TURTLES (CARETTA CARETTA).

Colloid osmotic pressure (COP) is an important component of the forces that manage capillary filtration and is determined by circulating plasma proteins. Patients affected by conditions resulting in hypoproteinemia often suffer severe hemodynamic derangements, including decreased COP. Because chronically debilitated sea turtles (CDT) present with severe hypoproteinemia, the objectives of this study were to 1) determine differences in plasma COP and blood analyte data (packed cell volume [PCV], sodium, chloride, plasma protein fractions) in CDT at admission compared with data from apparently healthy rehabilitated turtles at time of release (HRT) admitted from various stranding causes, and 2) to investigate correlations of COP with these selected blood analytes. COP, PCV, and most plasma protein fractions (excluding pre-albumin and γ-globulins) were significantly lower in CDT upon admission as compared with HRT. Sodium and chloride did not significantly differ between CDT and HRT. A significant increase was observed with PCV and all plasma protein fractions as COP increased. Of all protein fractions tested, albumin contributed the most toward COP (r2 = 0.88, P < 0.001). The results of this study suggest that COP is significantly lower in CDT as compared with HRT, providing insight into the complexity of this critical clinical condition and a small step in advancing the understanding of associated hemodynamic imbalances. Although COP analysis is not readily available as a diagnostic test, this preliminary baseline data suggests that additional research studies are warranted, given the potential for optimization of fluid therapy during rehabilitation of CDT.

Emerging Insights into Brevetoxicosis in Sea Turtles.

This review summarizes the current understanding of how brevetoxins, produced by Karenia brevis during harmful algal blooms, impact sea turtle health. Sea turtles may be exposed to brevetoxins through ingestion, inhalation, maternal transfer, and potentially absorption through the skin. Brevetoxins bind to voltage-gated sodium channels in the central nervous system, disrupting cellular function and inducing neurological symptoms in affected sea turtles. Moreover, the current evidence suggests a broader and longer-term impact on sea turtle health beyond what is seen during stranding events. Diagnosis relies on the detection of brevetoxins in tissues and plasma from stranded turtles. The current treatment of choice, intravenous lipid emulsion therapy, may rapidly reduce symptoms and brevetoxin concentrations, improving survival rates. Monitoring, prevention, and control strategies for harmful algal blooms are discussed. However, as the frequency and severity of blooms are expected to increase due to climate change and increased environmental pollution, continued research is needed to better understand the sublethal effects of brevetoxins on sea turtles and the impact on hatchlings, as well as the pharmacokinetic mechanisms underlying brevetoxicosis. Moreover, research into the optimization of treatments may help to protect endangered sea turtle populations in the face of this growing threat.

Blood analyte reference intervals and correlations with trace elements of immature and adult Eastern Pacific green turtles (Chelonia mydas) in coastal lagoons of Baja California Sur, México.

Sea turtles can bioaccumulate high concentrations of potentially toxic contaminants. To better understand trace element effects on sea turtles' health, we established reference intervals for hematological and plasma biochemical analytes in 40 in-water, foraging immature and adult Eastern Pacific green turtles (Chelonia mydas) from two coastal lagoons in Baja California Sur, quantified whole blood concentrations of eight trace elements, and assessed their correlations. Rank-order trace element concentrations in both immature and adult turtles was zinc > selenium > nickel > arsenic > copper > cadmium > lead > manganese. Immature turtles had significantly higher copper and lower nickel and zinc concentrations. Additionally, a number of relationships between trace elements and blood analytes were identified. These data provide baseline information useful for future investigations into this population, or in other geographic regions and various life-stage classes.

Morphometrics and blood analytes of leatherback sea turtle hatchlings (Dermochelys coriacea) from Florida: reference intervals, temporal trends with clutch deposition date, and body size correlations.

The northwest Atlantic leatherback sea turtle (Dermochelys coriacea) population is exhibiting decreasing trends along numerous nesting beaches. Since population health and viability are inherently linked, it is important to establish species- and life-stage class-specific blood analyte reference intervals (RIs) so that effects of future disturbances on organismal health can be better understood. For hatchling leatherbacks, the objectives of this study were to (1) establish RIs for morphometrics and blood analytes; (2) evaluate correlations between hatchling morphometrics, blood analytes, and hatching success; and (3) determine temporal trends in hatchling morphometrics and blood analytes across nesting season. Blood samples were collected from 176 naturally emerging leatherback hatchlings from 18 clutches. Reference intervals were established for morphometrics and blood analytes. Negative relationships were noted between hatchling mass and packed cell volume, total white blood cells, heterophils, lymphocytes, and total protein and between body condition index (BCI) and immature red blood cells (RBC), RBC polychromasia and anisocytosis, and total protein. Clutch deposition date showed positive relationships with lymphocytes and total protein, and negative relationships with hatchling mass and BCI. Hatching success was positively correlated with mass, and negatively with total protein and glucose, suggesting that nutritional provisions in eggs, incubation time, and/or metabolic rates could change later in the season and affect survivorship. These various observed correlations provide evidence for increased physiological stress (e.g., inflammation, subclinical dehydration) in hatchlings emerging later in nesting season, presumably due to increased nest temperatures or other environmental factors (e.g., moisture/rainfall). Data reported herein provide morphometric and blood analyte data for leatherback hatchlings and will allow for future investigations into spatiotemporal trends and responses to various stressors.

NONPIGMENTED VERSUS PIGMENTED: HEALTH VARIABLES AND GENETICS OF ALBINO FLORIDA GREEN TURTLE (CHELONIA MYDAS) HATCHLINGS COMPARED WITH NORMALLY PIGMENTED HATCHLINGS FROM THE SAME CLUTCH.

At the time of hatchling emergence from a nest laid on Juno Beach, Florida, US, by a normally pigmented green turtle (Chelonia mydas), 23 albino hatchlings and 75 normally pigmented hatchlings were observed. This condition is rarely seen in sea turtles, and little is known about blood analytes and genetics of albino wildlife to date. Therefore, the objective of our study was to assess and compare morphometric measurements (mass, minimum straight carapace length, body condition index), carapacial scute anomalies, a suite of hematologic and plasma biochemical analytes, and two glucose analysis methodologies (glucometer and dry chemistry analysis) in albino (n=20) versus normally pigmented (n=24) hatchlings from this nest. Genetic analyses were completed to identify paternal contributions of hatchlings and to test Mendelian inheritance assumptions. Although morphometric measurements, scute anomalies, and leukocyte morphology were similar between albino and normally pigmented hatchlings, several differences were observed in blood analyte data: immature erythrocytes, packed cell volume, heterophil:lymphocyte ratio, and glucose concentrations (by both methodologies) were significantly higher, whereas absolute immature heterophils, absolute lymphocytes, number of erythrocyte micronuclei, sodium, and chloride were significantly lower in albino hatchlings compared with normally pigmented hatchlings. Considerations for these differences include a stress response from sampling (e.g., timing of procedures or possibly from photosensitivity or reduced visual acuity in albinos) and different osmoregulation, which may reflect physiologic variations or stress. There was a small positive bias (0.10 mmol/L) with glucose by glucometer, similar to reports in other sea turtle species and confirming its suitability for use in hatchlings. All albino hatchlings analyzed (n=10) were from the same father, but the normally pigmented hatchlings (n=24) were from two other fathers. These findings provide insight into the physiology and genetics of albinism in sea turtles.

Morphologic and physiologic characteristics of green sea turtle (Chelonia mydas) hatchlings in southeastern Florida, USA.

The ability of sea turtle hatchlings to survive into adulthood is related, in part, to their individual health status. Documenting a variety of health data is essential for assessing individual and population health. In this study, we report health indices for 297 green sea turtle (Chelonia mydas) hatchlings that emerged from 32 nests deposited on Juno Beach, Florida, USA in June-July, 2017. Results of physical examination, morphometrics, and infectious disease testing (chelonid alphaherpesvirus 5, ChHV5), and blood analyte reference intervals (hematology, plasma protein, glucose) are presented. Carapacial scute abnormalities were observed in 36% (108/297) of all hatchlings, including abnormal vertebral (86/297, 29%), lateral (72/297, 24%), and both vertebral and lateral (50/297, 17%) scutes. Hatchlings from nests laid in July, which was ~ 1.6 °C warmer than June, had significantly shorter incubation periods, and higher body mass, straight carapace length, body condition index, packed cell volume, and heterophil:lymphocyte ratios compared to hatchlings from nests laid in June. These results suggest that incubation temperatures are linked to hatchling developmental factors and size, nutritional and/or hydration status, and/or blood cell dynamics. Blood samples from all 297 hatchlings tested negative for ChHV5 DNA via quantitative PCR, including 86 hatchlings from the nests of 11 adult females that tested positive for ChHV5 via qPCR or serology in a separate study, lending support to the hypothesis that ChHV5 is horizontally (rather than vertically) transmitted among green turtles. Information resulting from this study represents a useful dataset for comparison to future health assessment and population monitoring studies of green turtle hatchlings in the northwestern Atlantic Ocean.

Method Comparison of ß-Hydroxybutyrate Using a Point-of-Care Device and Dry Chemistry Analyzer in Three Sea Turtle Species.

The ketone ß-hydroxybutyrate (BHB) serves as an energy source when bodily energy stores are low. Concentrations of this blood analyte are often determined by spectrophotometric quantitative assays with a dry chemistry analyzer; however, rapid assessment with point-of-care devices have the potential to improve assessment of animals in the field or in clinical settings. We measured BHB concentrations in whole blood samples from 54 leatherback (Dermochelys coriacea), 27 loggerhead (Caretta caretta), and 14 green (Chelonia mydas) sea turtles in Florida, US with a point-of-care device and validated its use with corresponding plasma samples and dry chemistry analyzer as the gold standard. Concentrations of BHB highly correlated between the two methods for all three species, with loggerheads showing the best agreement and lowest bias. Therefore, the point-of-care device used for this study (Lucidplus ß-ketone monitoring system) is probably appropriate for sea turtle BHB measurements.

Cultivating epizoic diatoms provides insights into the evolution and ecology of both epibionts and hosts.

Our understanding of the importance of microbiomes on large aquatic animals-such as whales, sea turtles and manatees-has advanced considerably in recent years. The latest observations indicate that epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful approach to culture putatively obligate epizoic diatoms without their hosts. That some taxa can be cultured independently from their epizoic habitat raises several questions about the nature of the interaction between these animals and their epibionts. This insight allows us to propose further applications and research avenues in this growing area of study. Analyzing the DNA sequences of these cultured strains, we found that several unique diatom taxa have evolved independently to occupy epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Two heads are not always better than one: Craniofacial and axial bifurcation in cheloniid embryos and hatchlings (Chelonia mydas and Caretta caretta).

Morphological data on craniofacial and axial bifurcation in sea turtles is not well documented in the literature. Here, we use micro-computed tomography (µ-CT) imaging to describe the body, skull, and vertebral morphology in axially-bifurcated cheloniid sea turtle embryos and hatchlings (Chelonia mydas and Caretta caretta) from south Florida beaches. We describe three types of craniofacial and axial bifurcations: bifacial, bicephalic, and bicephalic with biaxial duplication ranging from facial bones to the sacrum. We predicted smaller body dimensions in bifacial and bicephalic embryos and hatchlings compared with their normal counterparts. In addition, we hypothesized that bicephalic individuals would have greater rostral deviation angles than bifacial animals, and that vertebral dimensions would vary between the control and anomalous embryos and hatchlings. Among hatchlings (developmental Stage 31), we found that maximum curved carapace length and curved carapace width were greatest in the control specimens when compared with the anomalous specimens. Overall, we found that rostral deviations were smaller in bifacial animals compared with their bicephalic counterparts. Right and left rostral deviations were symmetrical or nearly symmetrical in all bifacial and bicephalic specimens. Among C. caretta, we found that bicephalic animals had greater standardized vertebral measurements than their bifacial conspecifics. In bifacial animals, bifurcation extended to either the frontal or parietal skull bones, while duplication extended to C5 vertebrae and T8 vertebrae in bicephalic animals. This study provides an in-depth description of anatomical alterations associated with these abnormalities. Prognosis of these organisms is poor; however, understanding the prevalence of these malformations can allow for better assessments of population health, as numerous environmental factors are known to lead to these changes.

Use of intravenous lipid emulsion therapy as a novel treatment for brevetoxicosis in sea turtles.

The southwest coast of Florida experiences annual red tides, a type of harmful algal bloom that results from high concentrations of Karenia brevis. These dinoflagellates release lipophilic neurotoxins, known as brevetoxins, that bind to sodium channels and inhibit their inactivation, resulting in a variety of symptoms that can lead to mass sea turtle strandings. Traditional therapies for brevetoxicosis include standard and supportive care (SSC) and/or dehydration therapy; however, these treatments are slow-acting and often ineffective. Because red tide events occur annually in Florida, our objective was to test intravenous lipid emulsion (ILE) as a rapid treatment for brevetoxicosis in sea turtles and examine potential impacts on toxin clearance rates, symptom reduction, rehabilitation time, and survival rates. Sea turtles exhibiting neurological symptoms related to brevetoxicosis were brought to rehabilitation from 2018-2019. Upon admission, blood samples were collected, followed by immediate administration of 25 mg ILE/kg body mass (Intralipid® 20%) at 1 mL/min using infusion pumps. Blood samples were collected at numerous intervals post-ILE delivery and analyzed for brevetoxins using enzyme-linked immunosorbent assays. In total, nine (four subadults, one adult female, four adult males) loggerheads (Caretta caretta), five (four juvenile, one adult female) Kemp's ridleys (Lepidochelys kempii), and four juvenile green turtles (Chelonia mydas) were included in this study. We found that plasma brevetoxins declined faster compared to turtles that received only SSC. Additionally, survival rate of these patients was 94% (17/18), which is significantly higher than previous studies that used SSC and/or dehydration therapy (47%; 46/99). Nearly all symptoms were eliminated within 24-48 h, whereas using SSC, symptom elimination could take up to seven days or more. The dosage given here (25 mg/kg) was sufficient for turtles in this study, but the use of a higher dosage (50-100 mg/kg) for those animals experiencing severe symptoms may be considered. These types of fast-acting treatment plans are necessary for rehabilitation facilities that are already resource-limited. Intravenous lipid emulsion therapy has the potential to reduce rehabilitation time, save resources, and increase survival of sea turtles and other marine animals experiencing brevetoxicosis.

Plasma proteomics of green turtles (Chelonia mydas) reveals pathway shifts and potential biomarker candidates associated with health and disease.

Evaluating sea turtle health can be challenging due to an incomplete understanding of pathophysiologic responses in these species. Proteome characterization of clinical plasma samples can provide insights into disease progression and prospective biomarker targets. A TMT-10-plex-LC-MS/MS platform was used to characterize the plasma proteome of five, juvenile, green turtles (Chelonia mydas) and compare qualitative and quantitative protein changes during moribund and recovered states. The 10 plasma samples yielded a total of 670 unique proteins. Using ≥1.2-fold change in protein abundance as a benchmark for physiologic upregulation or downregulation, 233 (34.8%) were differentially regulated in at least one turtle between moribund and recovered states. Forty-six proteins (6.9%) were differentially regulated in all five turtles with two proteins (0.3%) demonstrating a statistically significant change. A principle component analysis showed protein abundance loosely clustered between moribund samples or recovered samples and for turtles that presented with trauma (n = 3) or as intestinal floaters (n = 2). Gene Ontology terms demonstrated that moribund samples were represented by a higher number of proteins associated with blood coagulation, adaptive immune responses and acute phase response, while recovered turtle samples included a relatively higher number of proteins associated with metabolic processes and response to nutrients. Abundance levels of 48 proteins (7.2%) in moribund samples significantly correlated with total protein, albumin and/or globulin levels quantified by biochemical analysis. Differentially regulated proteins identified with immunologic and physiologic functions are discussed for their possible role in the green turtle pathophysiologic response and for their potential use as diagnostic biomarkers. These findings enhance our ability to interpret sea turtle health and further progress conservation, research and rehabilitation programs for these ecologically important species.

Comparison of 2 glucose analytical methodologies in immature Kemp's ridley sea turtles: dry chemistry of plasma versus point-of-care glucometer analysis of whole blood.

Blood glucose measurements provide important diagnostic information regarding stress, disease, and nutritional status. Glucose analytical methodologies include dry chemistry analysis (DCA) of plasma and point-of-care (POC) glucometer analysis of whole blood; however, these 2 methods differ in cost, required sample volume, and processing time. Because POC glucometers use built-in equations based on features of mammalian blood to convert whole blood measurements to plasma equivalent units, obtained glucose data must be compared and validated using gold-standard chemistry analytical methodology in reptiles. For in-water, trawl-captured, immature Kemp's ridley sea turtles (Lepidochelys kempii) from Georgia, USA, we observed significant, positive agreement between the 2 glucose determination methods; however, the glucometer overestimated glucose concentrations by 1.4 mmol/L on average in comparison to DCA and produced a wider range of results. The discordance of these results suggests that POC glucometer glucose data should be interpreted in the context of methodology- and brand-specific reference intervals along with concurrent packed cell volume data.

Comprehensive health assessment and blood analyte reference intervals of gopher tortoises (Gopherus polyphemus) in southeastern FL, USA.

The gopher tortoise (Gopherus polyphemus), a keystone species, is declining throughout its geographic range. Lack of knowledge with respect to the potential infectious diseases present within wild populations creates a dilemma for wildlife biologists, conservationists and public policy makers. The objective of this study was to conduct a health assessment of two previously unstudied gopher tortoise aggregations located at two sites in southeastern FL. Samples were collected from 91 tortoises (48 adults, 35 juveniles, 8 hatchlings) captured at Florida Atlantic University's Harbor Branch Oceanographic Institute, in Fort Pierce, FL, USA in 2019, and Loggerhead Park in Juno Beach, FL, USA, during 2018-2019. Samples of blood, nasal swabs and oral/cloacal swabs were analyzed for hematology, plasma protein electrophoretic profiles and infectious disease testing including Mycoplasma spp. serology and polymerase chain reaction (PCR) assays for Ranavirus, Herpesvirus and Anaplasma spp. Hematological and plasma protein electrophoresis reference intervals are presented for adult and juvenile tortoises from both sites combined. Clinical signs consistent with upper respiratory tract disease (URTD) were observed in 18/91 (20%) tortoises, and antibodies to Mycoplasma agassizii were detected in 33/77 (42.9%) tortoises. Adult tortoises were significantly more likely than juveniles to have URTD clinical signs, and statistically significant, positive relationships were observed between the presence of antibodies to Mycoplasma spp. and carapace length, packed cell volume and plasma globulin concentrations. Anaplasma spp. inclusions were observed in 8/82 (10%) tortoises, but PCR detected Anaplasma sp. in 21/83 (25%) tortoises. Herpesvirus and Ranavirus were not detected in any blood or swab samples. This work contributes important baseline information on the health of gopher tortoises toward the southern end of the species' range.

Mercury offloading in gametes and potential adverse effects of high mercury concentrations in blood and tissues of Atlantic Goliath Grouper Epinephelus itajara in the southeastern United States.

Mercury (Hg) is a ubiquitous and non-essential heavy metal that is highly toxic to aquatic organisms. Few studies examine Hg and its effects on wild fish populations. Here, we investigated the potential effects of Hg exposure on a large and long-lived marine species of conservation concern, the vulnerable Atlantic Goliath Grouper Epinephelus itajara. Our objectives were (1) to measure Hg (methyl-Hg; and total Hg = combined methyl-Hg and inorganic-Hg) concentrations in whole blood (WB) and gametes (eggs and sperm); (2) to investigate the relationships between Hg concentrations in muscle and liver with WB and gametes; (3) to investigate the relationships between Hg concentrations in liver, muscle, and WB with hematological and plasma biochemical analytes; and (4) to investigate the relationship between liver Hg and pigmented macrophage aggregates in liver tissue sections. We found several lines of evidence for potential adverse effects on Goliath Grouper health and reproduction by high Hg concentrations in liver, muscle, WB, and gametes, including (1) Hg concentrations in all tissues and gametes were well above observable ranges of marine and freshwater fishes from experimental exposure studies; (2) gamete Hg concentrations were among the highest recorded in wild fishes, with seasonal patterns suggesting females offload significant amounts of Hg into their eggs during the spawning season; (3) Methyl-Hg was highest in muscle, followed by sperm, liver, eggs, and WB; (4) there were significant correlations between liver, muscle, and WB Hg concentrations with various blood analytes; (5) vitellogenin positively correlated with female liver methyl-Hg concentrations, and was abnormally high in males, suggesting direct endocrine effects; and (6) liver total Hg positively correlated with pigmented macrophage aggregate count and percent area. This work will help guide future research examining the potential adverse effects of Hg and its role as an additional stressor on wild fish populations.

Insights on Immune Function in Free-Ranging Green Sea Turtles (Chelonia mydas) with and without Fibropapillomatosis.

Chelonid alphaherpesviruses 5 and 6 (ChHV5 and ChHV6) are viruses that affect wild sea turtle populations. ChHV5 is associated with the neoplastic disease fibropapillomatosis (FP), which affects green turtles (Chelonia mydas) in panzootic proportions. ChHV6 infection is associated with lung-eye-trachea disease (LETD), which has only been observed in maricultured sea turtles, although antibodies to ChHV6 have been detected in free-ranging turtles. To better understand herpesvirus prevalence and host immunity in various green turtle foraging aggregations in Florida, USA, our objectives were to compare measures of innate and adaptive immune function in relation to (1) FP tumor presence and severity, and (2) ChHV5 and ChHV6 infection status. Free-ranging, juvenile green turtles (N = 45) were captured and examined for external FP tumors in Florida's Big Bend, Indian River Lagoon, and Lake Worth Lagoon. Blood samples were collected upon capture and analyzed for ChHV5 and ChHV6 DNA, antibodies to ChHV5 and ChHV6, in vitro lymphocyte proliferation using a T-cell mitogen (concanavalin A), and natural killer cell activity. Despite an overall high FP prevalence (56%), ChHV5 DNA was only observed in one individual, whereas 20% of turtles tested positive for antibodies to ChHV5. ChHV6 DNA was not observed in any animals and only one turtle tested positive for ChHV6 antibodies. T-cell proliferation was not significantly related to FP presence, tumor burden, or ChHV5 seroprevalence; however, lymphocyte proliferation in response to concanavalin A was decreased in turtles with severe FP (N = 3). Lastly, green turtles with FP (N = 9) had significantly lower natural killer cell activity compared to FP-free turtles (N = 5). These results increase our understanding of immune system effects related to FP and provide evidence that immunosuppression occurs after the onset of FP disease.

Fibropapillomatosis and Chelonid Alphaherpesvirus 5 Infection in Kemp's Ridley Sea Turtles (Lepidochelys kempii).

Fibropapillomatosis (FP), a debilitating, infectious neoplastic disease, is rarely reported in endangered Kemp's ridley sea turtles (Lepidochelys kempii). With this study, we describe FP and the associated chelonid alphaherpesvirus 5 (ChHV5) in Kemp's ridley turtles encountered in the United States during 2006-2020. Analysis of 22 case reports of Kemp's ridley turtles with FP revealed that while the disease was mild in most cases, 54.5% were adult turtles, a reproductively valuable age class whose survival is a priority for population recovery. Of 51 blood samples from tumor-free turtles and 12 tumor samples from turtles with FP, 7.8% and 91.7%, respectively, tested positive for ChHV5 DNA via quantitative polymerase chain reaction (qPCR). Viral genome shotgun sequencing and phylogenetic analysis of six tumor samples show that ChHV5 sequences in Kemp's ridley turtles encountered in the Gulf of Mexico and northwestern Atlantic cluster with ChHV5 sequences identified in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtles from Hawaii, the southwestern Atlantic Ocean, and the Caribbean. Results suggest an interspecific, spatiotemporal spread of FP among Kemp's ridley turtles in regions where the disease is enzootic. Although FP is currently uncommon in this species, it remains a health concern due to its uncertain pathogenesis and potential relationship with habitat degradation.

Harmful algal and cyanobacterial toxins in foraging green turtles (Chelonia mydas) in Florida's Big Bend.

Numerous toxin-producing harmful algal (HAB) species occur in Florida's coastal waters. Exposure to these toxins has been shown to have sublethal effects in sea turtles. The objective of this study was to establish concentrations of 10 HAB toxins in plasma samples from green turtles (Chelonia mydas) foraging in Florida's Big Bend. Domoic acid, lyngbyatoxin-A, microcystins, nodularin, and okadaic acid were detected, demonstrating exposure to these HAB toxins, which are also a public health concern.