Health and nutrition of loggerhead sea turtles (Caretta caretta) in the southeastern United States.

Loggerhead sea turtles (Caretta caretta) are opportunistic carnivores that feed primarily on benthic invertebrates and fish. Sea turtle rehabilitation requires provision of a species-specific, balanced diet that supplies nutrition similar to that of a wild diet; this can be challenging because free-ranging loggerheads' diets vary depending on their life stage and geographic location, with predominant prey species dictated by local availability. The goal of this study was to better understand the nutritional needs of subadult and adult loggerheads in rehabilitation. This was accomplished by conducting a retrospective survey of stomach contents identified during gross necropsy of 153 deceased loggerheads that stranded in coastal Georgia, USA. A total of 288 different forage items were identified; the most frequently observed prey items belong to the subphylum Crustacea (N = 131), followed by bony fish (Osteichthyes; N = 45), gastropod mollusks (N = 40), bivalve mollusks (N = 23), and Atlantic horseshoe crabs (Limulus polyphemus; N = 15). The proportions of certain prey items differed significantly with turtle size; adult turtles ate proportionately more gastropods (p = 0.001), and subadults ate proportionately more fish (p = 0.01). Stomach contents information was used to determine common local prey items (blue crab, cannonball jellyfish, horseshoe crab, whelk), which were evaluated for nutritional content. Additionally, we compared hematology and plasma biochemistry profiles (including proteins, trace minerals, and vitamins) between four cohorts of loggerhead turtles, including free-ranging subadults and adults, nesting females, and loggerheads undergoing rehabilitation. This information was applied to inform a regionally specific, formulated diet for tube feeding, and a supplement containing vitamins and minerals for captive loggerheads, to more closely approximate the nutritional content of their natural diet. Assessing the regional and temporal variability in loggerhead diets is an important component in their effective conservation because resultant data can be used to help understand the impacts of environmental perturbations on benthic food webs.

Baseline Health and Nutritional Parameters of Wild Sand Tigers Sampled in Delaware Bay.

Species-specific hematological reference values are essential for diagnosis and treatment of disease and maintaining overall health of animals. This information is lacking for many species of elasmobranchs maintained in zoos and aquaria, thus reducing the effectiveness of care for these animals. Descriptive statistics and reference intervals were calculated for hematocrit and complete blood cell counts, biochemistry and protein electrophoresis parameters, trace minerals, vitamins, heavy metals, reproductive hormones, and fatty acids in the blood of 153 wild Sand Tigers Carcharias taurus of both sexes and a range of sizes caught in Delaware Bay (Delaware, USA). Mean hematocrit, total white blood cell counts, lymphocyte differentials, glucose, phosphorus, amylase, and aspartate aminotransferase levels were significantly higher in juveniles than in adults. Levels of estradiol, progesterone, testosterone, and differences in selenium and eicosapentaenoic acid (a polyunsaturated fatty acid) between males and females suggest that they are important parameters for improving Sand Tiger breeding success in managed care. Finally, blood metal levels for arsenic, cadmium, lead, and mercury suggest low levels of contaminant exposure for Sand Tigers during their summer residence in Delaware Bay. The results of this study provide baseline health parameters for wild Sand Tigers that will aid in effective maintenance of aquarium animals and contribute to a greater understanding of the biology of these sharks and efforts to accomplish sustainable management of their populations.

COMPARISON OF HEMATOLOGICAL, PLASMA BIOCHEMICAL, AND NUTRITIONAL ANALYTES OF REHABILITATING AND APPARENTLY HEALTHY FREE-RANGING ATLANTIC GREEN TURTLES (CHELONIA MYDAS).

Green turtles (Chelonia mydas) are unique because hatchlings and pelagic juveniles are carnivorous, whereas later life stages become primarily herbivorous. Although this dietary shift is well understood, nutritional requirements at each developmental stage are not. Diet selection during rehabilitation is challenging, because turtles are often fed high-protein, low-fiber diets to counteract poor appetite and emaciation, which can result in gastrointestinal disorders. The objective of this study was to analyze hematology, plasma biochemistry, and nutritional analytes to determine the effect of diet on rehabilitating green turtle health and recovery. Turtles in rehabilitation at the Georgia Sea Turtle Center (GSTC) on Jekyll Island, Georgia (n = 21) were sampled at admission, mid-rehabilitation, and recovery. Duration of rehabilitation ranged from 48 to 233 days (mean = 117) and included a shift from a mixed seafood-vegetable diet at admission to a primarily herbivorous diet at recovery. For comparison, free-ranging turtles captured during an ongoing monitoring project in St. Lucie County, Florida (n = 34) were evaluated for the same variables. Several analytes improved during rehabilitation, including total protein, uric acid, potassium, and vitamins A and E. Others were significantly different, including higher cholesterol and triglycerides, but lower calcium, vitamin D, and magnesium in recovery compared with freeranging turtles. These variations illustrate the benefits of appropriate supportive care during rehabilitation as well as the importance of a species-specific diet for green turtles, and may facilitate formulation of nutritionally complete gel diets for this species under human care.

MEDICAL MANAGEMENT OF COELOMIC DISTENTION, ABNORMAL SWIMMING, SUBSTRATE RETENTION, AND HEMATOLOGIC CHANGES IN A REEF MANTA RAY (MANTA ALFREDI).

An adult, female, reef manta ray (Manta alfredi) was evaluated for abnormal swimming and coelomic distention after colliding with a floating dock in an exhibit. Initial clinical signs included abnormal posture and swimming. Clinical signs progressed to include muscle wasting, poor body condition, and coelomic distention. Further diagnostics revealed excessive exhibit substrate within the gastrointestinal tract, gastric ulcers, free coelomic fluid, possible spinal lesion, possible uterine abnormality, and hematologic changes. The ray was treated with repeated gastroscopic examinations to remove substrate from the stomach. Psyllium and mineral oil were administered in gel food to assist with passage of substrate through the gastrointestinal tract. Gastric ulcers were treated with sucralfate. Vitamin B complex, iron dextran, and Yunnan Paiyao were used to treat the anemia. Amikacin and sulfadimethoxine-ormetoprim were administered for suspected bacterial and coccidial infections, respectively. Over the course of 11 mo the ray returned to normal health.

The microbiome of African penguins (Spheniscus demersus) under managed care resembles that of wild marine mammals and birds.

Animals under managed care in zoos and aquariums are ideal surrogate study subjects for endangered species that are difficult to obtain in the wild. We compared the fecal and oral microbiomes of healthy, managed African penguins (Spheniscus demersus) to those of other domestic and wild vertebrate hosts to determine how host identity, diet, and environment shape the penguin microbiome. The African penguin oral microbiome was more similar to that of piscivorous marine mammals, suggesting that diet and a marine environment together play a strong role in shaping the oral microbiome. Conversely, the penguin cloaca/fecal microbiome was more similar to that of other birds, suggesting that host phylogeny plays a significant role in shaping the gut microbiome. Although the penguins were born under managed care, they had a gut microbiome more similar to that of wild bird species compared to domesticated (factory-farmed) birds, suggesting that the managed care environment and diet resemble those experienced by wild birds. Finally, the microbiome composition at external body sites was broadly similar to that of the habitat, suggesting sharing of microbes between animals and their environment. Future studies should link these results to microbial functional capacity and host health, which will help inform conservation efforts.

An Acute Mortality Event Associated with Novel Macrorhabdus ornithogaster Infection and Underlying Factors in a Newly-Established Captive Group of American White Ibis (Eudocimus albus) Nestlings.

Twenty-four American white ibis (Eudocimus albus) nestlings were collected in Florida (USA) on 17 April 2017 to establish a captive flock. On 7 May 2017, three birds died suddenly, following severe lethargy, hemorrhaging from the mouth and nares, anorexia, and production of bright-green colored feces. An additional ibis with delayed growth and pathological fractures was euthanized 18 May 2017. Severe ventriculitis associated with Macrorhabdus ornithogaster was noted in all four birds, bacterial sepsis was confirmed in one bird by culture and histologic examination, and bacterial endotoxemia was suspected in two birds based on gross and histologic examination, but no bacteria were isolated from these birds. Birds also had vitamin E liver levels consistent with coagulopathy previously described in pelicans. We sampled feces from 91 adult, free-living, healthy ibis in Florida in July 2017 and found 71% were shedding organisms with morphologic characteristics consistent with Macrorhabdus sp. Molecular characterization of the ibis-origin M. ornithogaster showed it was phylogenetically related to numerous M. ornithogaster sequences. It is unknown if M. ornithogaster infection resulted in clinical disease as a result of dietary or stress-related dysbiosis, or other factors. Macrorhabdus-associated disease has not previously been confirmed in wading birds. We discuss potential associations of gastric M. ornithogaster infection with morbidity and mortality in these cases and highlight the need for additional studies on this pathogen in free-living birds.

Associations between total mercury, trace minerals, and blood health markers in Northwest Atlantic white sharks (Carcharodon carcharias).

The ecology and life-histories of white sharks make this species susceptible to mercury bioaccumulation; however, the health consequences of mercury exposure are understudied. We measured muscle and plasma total mercury (THg), health markers, and trace minerals in Northwest Atlantic white sharks. THg in muscle tissue averaged 10.0 mg/kg dry weight, while THg in blood plasma averaged 533 µg/L. THg levels in plasma and muscle were positively correlated with shark precaudal length (153-419 cm), and THg was bioaccumulated proportionally in muscle and plasma. Nine sharks had selenium:mercury molar ratios in blood plasma >1.0, indicating that for certain individuals the potential protective effects of the trace mineral were diminished, whereas excess selenium may have protected other individuals. No relationships between plasma THg and any trace minerals or health markers were identified. Thus, we found no evidence of negative effects of Hg bioaccumulation, even in sharks with very high THg.

Microbiome differences between wild and aquarium whitespotted eagle rays (Aetobatus narinari).

BACKGROUND: Animal-associated microbiomes can be influenced by both host and environmental factors. Comparing wild animals to those in zoos or aquariums can help disentangle the effects of host versus environmental factors, while also testing whether managed conditions foster a 'natural' host microbiome. Focusing on an endangered elasmobranch species-the whitespotted eagle ray Aetobatus narinari-we compared the skin, gill, and cloaca microbiomes of wild individuals to those at Georgia Aquarium. Whitespotted eagle ray microbiomes from Georgia Aquarium were also compared to those of cownose rays (Rhinoptera bonasus) in the same exhibit, allowing us to explore the effect of host identity on the ray microbiome. RESULTS: Long-term veterinary monitoring indicated that the rays in managed care did not have a history of disease and maintained health parameters consistent with those of wild individuals, with one exception. Aquarium whitespotted eagle rays were regularly treated to control parasite loads, but the effects on animal health were subclinical. Microbiome α- and ß-diversity differed between wild versus aquarium whitespotted eagle rays at all body sites, with α-diversity significantly higher in wild individuals. ß-diversity differences in wild versus aquarium whitespotted eagle rays were greater for skin and gill microbiomes compared to those of the cloaca. At each body site, we also detected microbial taxa shared between wild and aquarium eagle rays. Additionally, the cloaca, skin, and gill microbiomes of aquarium eagle rays differed from those of cownose rays in the same exhibit. Potentially pathogenic bacteria were at low abundance in all wild and aquarium rays. CONCLUSION: For whitespotted eagle rays, managed care was associated with a microbiome differing significantly from that of wild individuals. These differences were not absolute, as the microbiome of aquarium rays shared members with that of wild counterparts and was distinct from that of a cohabitating ray species. Eagle rays under managed care appear healthy, suggesting that their microbiomes are not associated with compromised host health. However, the ray microbiome is dynamic, differing with both environmental factors and host identity. Monitoring of aquarium ray microbiomes over time may identify taxonomic patterns that co-vary with host health.

Microbiome structure in large pelagic sharks with distinct feeding ecologies.

BACKGROUND: Sharks play essential roles in ocean food webs and human culture, but also face population declines worldwide due to human activity. The relationship between sharks and the microbes on and in the shark body is unclear, despite research on other animals showing the microbiome as intertwined with host physiology, immunity, and ecology. Research on shark-microbe interactions faces the significant challenge of sampling the largest and most elusive shark species. We leveraged a unique sampling infrastructure to compare the microbiomes of two apex predators, the white (Carcharodon carcharias) and tiger shark (Galeocerdo cuvier), to those of the filter-feeding whale shark (Rhincodon typus), allowing us to explore the effects of feeding mode on intestinal microbiome diversity and metabolic function, and environmental exposure on the diversity of microbes external to the body (on the skin, gill). RESULTS: The fecal microbiomes of white and whale sharks were highly similar in taxonomic and gene category composition despite differences in host feeding mode and diet. Fecal microbiomes from these species were also taxon-poor compared to those of many other vertebrates and were more similar to those of predatory teleost fishes and toothed whales than to those of filter-feeding baleen whales. In contrast, microbiomes of external body niches were taxon-rich and significantly influenced by diversity in the water column microbiome. CONCLUSIONS: These results suggest complex roles for host identity, diet, and environmental exposure in structuring the shark microbiome and identify a small, but conserved, number of intestinal microbial taxa as potential contributors to shark physiology.

Elucidating shark diets with DNA metabarcoding from cloacal swabs.

Animal dietary information provides the foundation for understanding trophic relationships, which is essential for ecosystem management. Yet, in marine systems, high-resolution diet reconstruction tools are currently under-developed. This is particularly pertinent for large marine vertebrates, for which direct foraging behaviour is difficult or impossible to observe and, due to their conservation status, the collection of stomach contents at adequate sample sizes is frequently impossible. Consequently, the diets of many groups, such as sharks, have largely remained unresolved. To address this knowledge gap, we applied metabarcoding to prey DNA in faecal residues (fDNA) collected on cotton swabs from the inside of a shark's cloaca. We used a previously published primer set targeting a small section of the 12S rRNA mitochondrial gene to amplify teleost prey species DNA. We tested the utility of this method in a controlled feeding experiment with captive juvenile lemon sharks (Negaprion brevirostris) and on free-ranging juvenile bull sharks (Carcharhinus leucas). In the captive trial, we successfully isolated and correctly identified teleost prey DNA without incurring environmental DNA contamination from the surrounding seawater. In the field, we were able to reconstruct high-resolution teleost dietary information from juvenile C. leucas fDNA that was generally consistent with expectations based on published diet studies of this species. While further investigation is needed to validate the method for larger sharks and other species, it is expected to be broadly applicable to aquatic vertebrates and provides an opportunity to advance our understanding of trophic interactions in marine and freshwater systems.

Elasmobranch microbiomes: emerging patterns and implications for host health and ecology.

Elasmobranchs (sharks, skates and rays) are of broad ecological, economic, and societal value. These globally important fishes are experiencing sharp population declines as a result of human activity in the oceans. Research to understand elasmobranch ecology and conservation is critical and has now begun to explore the role of body-associated microbiomes in shaping elasmobranch health. Here, we review the burgeoning efforts to understand elasmobranch microbiomes, highlighting microbiome variation among gastrointestinal, oral, skin, and blood-associated niches. We identify major bacterial lineages in the microbiome, challenges to the field, key unanswered questions, and avenues for future work. We argue for prioritizing research to determine how microbiomes interact mechanistically with the unique physiology of elasmobranchs, potentially identifying roles in host immunity, disease, nutrition, and waste processing. Understanding elasmobranch-microbiome interactions is critical for predicting how sharks and rays respond to a changing ocean and for managing healthy populations in managed care.

Seasonal patterns in stable isotope and fatty acid profiles of southern stingrays (Hypanus americana) at Stingray City Sandbar, Grand Cayman.

Ecotourism opportunities in the marine environment often rely heavily on provisioning to ensure the viewing of cryptic species by the public. However, intentional feeding of wildlife can impact numerous aspects of an animals' behavior and ecology. Southern stingrays (Hypanus americana) provisioned at Stingray City Sandbar (SCS) in Grand Cayman have altered diel activity patterns and decreased measures of health. This study looked at seasonal changes in stable isotope (SI) and fatty acid (FA) profiles of provisioned stingrays at SCS. Plasma δ15N was higher in male stingrays (11.86 ± 1.71‰) compared to females (10.70 ± 1.71‰). Lower values for δ15N in males and females were measured in October during low tourist season, suggesting stingrays may be forced to rely on native prey items to supplement the decreased amount of provisioned squid available during this time. Plasma FA profiles were significantly different between sexes and across sampling time points, with FAs 22:6n3, 16:0, 20:5n3, 18:1n3C, 18:0 and 18:1n9T contributing to dissimilarity scores between groups. Dietary FAs primarily contributed to differences between males and females lending further evidence to differences in foraging patterns at SCS, likely due to intraspecific competition. Further, canonical analysis of principal coordinates (CAP) analysis of FA profiles suggest similar diets during peak tourist season and differences in diet between males and females during the low season. This study demonstrates alterations in feeding ecology in stingrays at SCS which is of critical importance for effective management of the SCS aggregation.

The effect of diet on the gastrointestinal microbiome of juvenile rehabilitating green turtles (Chelonia mydas).

Threatened and endangered green turtles (Chelonia mydas) are unique because as juveniles they recruit from pelagic to near-shore waters and shift from an omnivorous to primarily herbivorous diet (i.e. seagrass and algae). Nevertheless, when injured and ill animals are admitted to rehabilitation, animal protein (e.g. seafood) is often offered to combat poor appetite and emaciation. We examined how the fecal microbiome of juvenile green turtles changed in response to a dietary shift during rehabilitation. We collected fecal samples from January 2014 -January 2016 from turtles (N = 17) in rehabilitation at the Georgia Sea Turtle Center and used next generation sequencing to analyze bacterial community composition. Samples were collected at admission, mid-rehabilitation, and recovery, which entailed a shift from a mixed seafood-vegetable diet at admission to a primarily herbivorous diet at recovery. The dominant phyla changed over time, from primarily Firmicutes (55.0%) with less Bacteroidetes (11.4%) at admission, to primarily Bacteroidetes (38.4%) and less Firmicutes (31.8%) at recovery. While the microbiome likely shifts with the changing health status of individuals, this consistent inversion of Bacteroidetes and Firmicutes among individuals likely reflects the increased need for protein digestion, for which Bacteroidetes are important. Firmicutes are significant in metabolizing plant polysaccharides; thus, fewer Firmicutes may result in underutilization of wild diet items in released individuals. This study demonstrates the importance of transitioning rehabilitating green turtles to an herbivorous diet as soon as possible to afford them the best probability of survival.

No evidence of metabolic depression in Western Alaskan juvenile Steller sea lions (Eumetopias jubatus).

Steller sea lion (Eumetopias jubatus) populations have undergone precipitous declines through their western Alaskan range over the last four decades with the leading hypothesis to explain this decline centering around changing prey quality, quantity, or availability for this species (i.e., nutritional stress hypothesis). Under chronic conditions of reduced food intake sea lions would conserve energy by limiting energy expenditures through lowering of metabolic rate known as metabolic depression. To examine the potential for nutritional stress, resting metabolic rate (RMR) and body composition were measured in free-ranging juvenile Steller sea lions (N = 91) at three distinct geographical locations (Southeast Alaska, Prince William Sound, Central Aleutian Islands) using open-flow respirometry and deuterium isotope dilution, respectively. Average sea lion RMR ranged from 6.7 to 36.2 MJ d(-1) and was influenced by body mass, total body lipid, and to a lesser extent, ambient air temperature and age. Sea lion pups captured in the Aleutian Islands (region of decline) had significantly greater body mass and total body lipid stores when compared to pups from Prince William Sound (region of decline) and Southeast Alaska (stable region). Along with evidence of robust body condition in Aleutian Island pups, no definitive differences were detected in RMR between sea lions sampled between eastern and western populations that could not be accounted for by higher percent total body lipid content, suggesting that that at the time of this study, Steller sea lions were not experiencing metabolic depression in the locations studied.

Thermal and digestive constraints to foraging behaviour in marine mammals.

While foraging models of terrestrial mammals are concerned primarily with optimizing time/energy budgets, models of foraging behaviour in marine mammals have been primarily concerned with physiological constraints. This has historically centred on calculations of aerobic dive limits. However, other physiological limits are key to forming foraging behaviour, including digestive limitations to food intake and thermoregulation. The ability of an animal to consume sufficient prey to meet its energy requirements is partly determined by its ability to acquire prey (limited by available foraging time, diving capabilities and thermoregulatory costs) and process that prey (limited by maximum digestion capacity and the time devoted to digestion). Failure to consume sufficient prey will have feedback effects on foraging, thermoregulation and digestive capacity through several interacting avenues. Energy deficits will be met through catabolism of tissues, principally the hypodermal lipid layer. Depletion of this blubber layer can affect both buoyancy and gait, increasing the costs and decreasing the efficiency of subsequent foraging attempts. Depletion of the insulative blubber layer may also increase thermoregulatory costs, which will decrease the foraging abilities through higher metabolic overheads. Thus, an energy deficit may lead to a downward spiral of increased tissue catabolism to pay for increased energy costs. Conversely, the heat generated through digestion and foraging activity may help to offset thermoregulatory costs. Finally, the circulatory demands of diving, thermoregulation and digestion may be mutually incompatible. This may force animals to alter time budgets to balance these exclusive demands. Analysis of these interacting processes will lead to a greater understanding of the physiological constraints within which the foraging behaviour must operate.