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.

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.

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.

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.

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.

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.

Fibropapillomatosis is associated with chelonid alphaherpesvirus 5 (ChHV5) and tumor formation in sea turtles. We collected blood samples from 113 green (Chelonia mydas) and 112 loggerhead (Caretta caretta) turtles without fibropapillomatosis, including 46 free-ranging turtles (20 green turtles, 26 loggerheads), captured in Core Sound, North Carolina, and 179 turtles (93 green turtles, 86 loggerheads) in rehabilitative care in North Carolina. Blood samples were analyzed for ChHV5 DNA using quantitative polymerase chain reaction (qPCR), and for antibodies to ChHV5 peptides using an enzyme-linked immunosorbent assay (ELISA). None of the samples from foraging turtles tested positive for ChHV5 by qPCR; ELISA was not used for foraging turtles. Samples from 18/179 (10.1%) rehabilitating turtles tested positive for ChHV5 using qPCR, and 32/56 (57.1%) rehabilitating turtles tested positive for antibodies to ChHV5 using ELISA. Five turtles that tested positive by qPCR or ELISA at admission converted to being undetectable during rehabilitation, and five that initially tested negative converted to being positive. Both sea turtle species were significantly more likely to test positive for ChHV5 using ELISA than with qPCR (p < 0.001). There was no difference in the proportions of green turtles versus loggerheads that tested positive for ChHV5 using qPCR, but loggerheads were significantly more likely than green turtles to test positive for ChHV5 using ELISA. This finding suggests that loggerheads infected with ChHV5 at some point in their life may be more able than green turtles to mount an effective immune response against recrudescent infection, pointing to species-specific genetic differences in the two species' immune response to ChHV5 infection. This is the first study to analyze antibodies to ChHV5 in loggerhead turtles and represents the most complete dataset on ChHV5 DNA detection in sea turtles encountered in the more northern latitudes of their western Atlantic habitat.

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.

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.

Natural biotoxins and anthropogenic toxicants pose a significant risk to sea turtle health. Documented effects of contaminants include potential disease progression and adverse impacts on development, immune function, and survival in these imperiled species. The shallow seagrass habitats of Florida's northwest coast (Big Bend) serve as an important developmental habitat for Kemp's ridley (Lepidochelys kempii) and green (Chelonia mydas) sea turtles; however, few studies have been conducted in this area. Our objectives were (1) to evaluate plasma analytes (mass, minimum straight carapace length, body condition index [BCI], fibropapilloma tumor score, lysozyme, superoxide dismutase, reactive oxygen/nitrogen species, plasma protein electrophoresis, cholesterol, and total solids) in Kemp's ridleys and green turtles and their correlation to brevetoxins that were released from a red tide bloom event from July-October 2014 in the Gulf of Mexico near Florida's Big Bend, and (2) to analyze red blood cells in Kemp's ridleys and green turtles for toxic elements (arsenic, cadmium, lead, mercury, selenium, thallium) with correlation to the measured plasma analytes. Positive correlations were observed between brevetoxins and α2-globulins in Kemp's ridleys and α2- and γ-globulins in green turtles, indicating potential immunostimulation. Arsenic, cadmium, and lead positively correlated with superoxide dismutase in Kemp's ridleys, suggesting oxidative stress. Lead and mercury in green turtles negatively correlated with BCI, while mercury positively correlated with total tumor score of green turtles afflicted with fibropapillomatosis, suggesting a possible association with mercury and increased tumor growth. The total tumor score of green turtles positively correlated with total protein, total globulins, α2-globulins, and γ-globulins, further suggesting inflammation and immunomodulation as a result of fibropapillomatosis. Lastly, brevetoxin concentrations were positively related to tumor score, indicating potential tumor promotion by brevetoxin. These results signify that brevetoxins and toxic elements elicit various negative effects on sea turtle health, including immune function, oxidative stress, and possibly disease progression.