Dolphin Samples Research Articles

First evidence of microplastic inhalation among free-ranging small cetaceans.

Plastic is a ubiquitous environmental contaminant, resulting in widespread exposure across terrestrial and marine spaces. In the environment, plastics can degrade into microparticles where exposure has been documented in a variety of fauna at all trophic levels. Human epidemiological studies have found relationships between inhaled microplastics and oxidative stress and inflammation. Previous studies of bottlenose dolphins (Tursiops truncatus) have reported prevalent exposure to plasticizing chemicals (e.g., phthalates) as well as particle loads in gastrointestinal tracts, but exposure from inhalation has not yet been studied. The objective of this study was to determine if inhalation is a viable route of microplastic exposure for free-ranging dolphins. Exhalation samples were opportunistically collected from dolphins residing in Sarasota Bay, Florida (n = 5) and Barataria Bay, Louisiana (n = 6) during catch-and-release health assessments to screen for microplastic particles. All dolphin samples contained at least one suspected microplastic particle, and polymer composition was determined for 100% of a subset (n = 17) of samples. Additional studies are warranted to better understand the extent of inhaled microplastics, as well as to explore impacts, given potential risks to lung function and health.

Bioaccumulation of trace elements in marine mammals: New data and transplacental transfer on threatened species

Coastal areas are affected by urban, industrial and agriculture pollutants runoff, wastewater and stormwater discharges, making this environment the final repository of chemical contaminants. These contaminants have the potential to spread out to the entire food chain, impacting marine life and the quality of their habitat. In this aspect, the concept of marine mammals as bioindicators provides an approach to the degree of contamination in the environment and to the identification and management of multiple sources of contaminants. The present study analyzed several elements like As, Ba, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, V and Zn in liver tissue from two dolphin species: Sotalia guianensis, a near-threatened species, and the vulnerable Pontoporia blainvillei. In the study, we also investigated if dolphins (population) recorded using the heaviest urban areas have higher concentrations of contaminants in their tissues. Dolphin samples (n = 40 S. guianensis; n = 97 P. blainvillei) were collected by daily monitoring carried out by Santos Basin Beach Monitoring Project (PMP-BS), from stranded individuals found in São Paulo state. The Spearman's rank correlation showed distinct correlations in the accumulation of trace elements by both species, indicating different sources of exposure to the elements studied or distinct biochemical processes between species. Interspecific and intraspecific variations were observed, possibly related to the individual distribution and feeding habits. Correlations were observed between age and concentrations of trace elements, positive for Cd, Hg and Mo. Finally, our findings indicate high levels of Cu, Zn, and concentrations of As, V and Hg in fetuses, in particular, an analysis was performed on a fetus found inside a stranded individual, indicating placental transfer as the first route of exposure for some elements.

Validating a Non-Invasive Method for Assessing Cortisol Concentrations in Scraped Epidermal Skin from Common Bottlenose Dolphins and Belugas.

Society is showing a growing concern about the welfare of cetaceans in captivity as well as cetaceans in the wild threatened by anthropogenic disturbances. The study of the physiological stress response is increasingly being used to address cetacean conservation and welfare issues. Within it, a newly described technique of extracting cortisol from epidermal desquamation may serve as a non-invasive, more integrated measure of a cetacean's stress response and welfare. However, confounding factors are common when measuring glucocorticoid hormones. In this study, we validated a steroid hormone extraction protocol and the use of a commercial enzyme immunoassay (EIA) test to measure cortisol concentrations in common bottlenose dolphin (Tursiops truncatus) and beluga (Delphinapterus leucas) epidermal samples. Moreover, we examined the effect of sample mass and body location on cortisol concentrations. Validation tests (i.e., assay specificity, accuracy, precision, and sensitivity) suggested that the method was suitable for the quantification of cortisol concentrations. Cortisol was extracted from small samples (0.01 g), but the amount of cortisol detected and the variability between duplicate extractions increased as the sample mass decreased. In common bottlenose dolphins, epidermal skin cortisol concentrations did not vary significantly across body locations while there was a significant effect of the individual. Overall, we present a contribution towards advancing and standardizing epidermis hormone assessments in cetaceans.

Novel processing and staining methodology of bottlenose dolphin (Tursiops truncatus) teeth for age determination

ABSTRACT Age determination of bottlenose dolphins (Tursiops truncatus) is a critical tool in understanding both individual and population health. There are many methods of aging bottlenose dolphins including analysis of teeth, pectoral flipper radiographs, and epigenetics. The most common and oldest method for aging toothed cetaceans is the counting of growth layer groups (GLGs) in the teeth. Current techniques have technical and repeatability challenges. Therefore, a processing technique that results in better resolution of GLGs is needed. This study compares different decalcifications and different histochemical staining techniques. Decalcification was done using 10% EDTA, Kristensen’s decalcification, and Rapid Decalcification Solution (RDO). Following decalcification and routine processing, GLGs were assessed using Hematoxylin and Eosin (H&E), hematoxylin, Giemsa, Wright-Giemsa, Toluidine Blue (T-Blue), Masson’s Trichrome, and Congo Red staining techniques. Decalcification with Kristensen’s and staining with Masson’s Trichrome and Congo Red were determined to best highlight GLGs. This processing and staining was then applied to a sample population of 102 bottlenose dolphins that were evaluated independently and blindly by two observers. Of the 102 dolphin samples, 13 (12.7%) were unable to age due to no clear distinction or distortion between GLGs.

Plastic, It's What's for Dinner: A Preliminary Comparison of Ingested Particles in Bottlenose Dolphins and Their Prey.

Microplastic ingestion was reported for common bottlenose dolphins (Tursiops truncatus) inhabiting Sarasota Bay, FL, USA, a community that also has prevalent exposure to plasticizers (i.e., phthalates) at concentrations higher than human reference populations. Exposure sources are currently unknown, but plastic-contaminated prey could be a vector. To explore the potential for trophic exposure, prey fish muscle and gastrointestinal tract (GIT) tissues and contents were screened for suspected microplastics, and particle properties (e.g., color, shape, surface texture) were compared with those observed in gastric samples from free-ranging dolphins. Twenty-nine fish across four species (hardhead catfish, Ariopsis felis; pigfish, Orthopristis chrysoptera; pinfish, Lagodon rhomboides; and Gulf toadfish, Opsanus beta) were collected from Sarasota Bay during September 2022. Overall, 97% of fish (n = 28) had suspected microplastics, and GIT abundance was higher than muscle. Fish and dolphin samples contained fibers and films; however, foams were common in dolphin samples and not observed in fish. Suspected tire wear particles (TWPs) were not in dolphin samples, but 23.1% and 32.0% of fish muscle and GIT samples, respectively, contained at least one suspected TWP. While some similarities in particles were shared between dolphins and fish, small sample sizes and incongruent findings for foams and TWPs suggest further investigation is warranted to understand trophic transfer potential.

Non-invasive age estimation based on faecal DNA using methylation-sensitive high-resolution melting for Indo-Pacific bottlenose dolphins.

Age is necessary information for the study of life history of wild animals. A general method to estimate the age of odontocetes is counting dental growth layer groups (GLGs). However, this method is highly invasive as it requires the capture and handling of individuals to collect their teeth. Recently, the development of DNA-based age estimation methods has been actively studied as an alternative to such invasive methods, of which many have relied on used biopsy samples. However, if DNA-based age estimation can be developed from faecal samples, age estimation can be performed entirely non-invasively. We developed an age estimation model using the methylation rate of two gene regions, GRIA2 and CDKN2A, measured through methylation-sensitive high-resolution melting (MS-HRM) from faecal samples of wild Indo-Pacific bottlenose dolphins (Tursiops aduncus). The age of individuals was known through conducting longitudinal individual identification surveys underwater. Methylation rates were quantified from 36 samples collected from 30 individuals. Both gene regions showed a significant correlation between age and methylation rate. The age estimation model was constructed based on the methylation rates of both genes which achieved sufficient accuracy (after LOOCV: MAE = 5.08, R2 = 0.33) for the ecological studies of the Indo-Pacific bottlenose dolphins, with a lifespan of 40-50 years. This is the first study to report the use of non-invasive faecal samples to estimate the age of marine mammals.

Comparison of de novo assembly using long-read shotgun metagenomic sequencing of viruses in fecal and serum samples from marine mammals.

Viral diseases of marine mammals are difficult to study, and this has led to a limited knowledge on emerging known and unknown viruses which are ongoing threats to animal health. Viruses are the leading cause of infectious disease-induced mass mortality events among marine mammals. In this study, we performed viral metagenomics in stool and serum samples from California sea lions (Zalophus californianus) and bottlenose dolphins (Tursiops truncates) using long-read nanopore sequencing. Two widely used long-read de novo assemblers, Canu and Metaflye, were evaluated to assemble viral metagenomic sequencing reads from marine mammals. Both Metaflye and Canu assembled similar viral contigs of vertebrates, such as Parvoviridae, and Poxviridae. Metaflye assembled viral contigs that aligned with one viral family that was not reproduced by Canu, while Canu assembled viral contigs that aligned with seven viral families that was not reproduced by Metaflye. Only Canu assembled viral contigs from dolphin and sea lion fecal samples that matched both protein and nucleotide RefSeq viral databases using BLASTx and BLASTn for Anelloviridae, Parvoviridae and Circoviridae families. Viral contigs assembled with Canu aligned with torque teno viruses and anelloviruses from vertebrate hosts. Viruses associated with invertebrate hosts including densoviruses, Ambidensovirus, and various Circoviridae isolates were also aligned. Some of the invertebrate and vertebrate viruses reported here are known to potentially cause mortality events and/or disease in different seals, sea stars, fish, and bivalve species. Canu performed better by producing the most viral contigs as compared to Metaflye with assemblies aligning to both protein and nucleotide databases. This study suggests that marine mammals can be used as important sentinels to surveil marine viruses that can potentially cause diseases in vertebrate and invertebrate hosts.

Application of in-source fragmentation to the identification of perfluoropentanoic acid and perfluorobutanoic acid in environmental matrices and human serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is the most common analytical technique for per- and polyfluoroalkyl substances (PFASs) research and monitoring. The PFAS identification requires a minimum of two multiple reaction monitoring (MRM) transition ions as quantifier transition ion and qualifier transition ion, respectively. The second transition ion abundance for perfluoropentanoic acid (PFPeA) and perfluobutanoic acid (PFBA) is too low for practical use. A method using the in-source fragment ions as the precursor ion for MRM or pseudo-MRM has been developed and evaluated for PFPeA and PFBA identification in various environmental abiotic and biotic samples including water, soil, sediment, WWTP sludge, fruits, vegetables, egg, macrophytes, fish, dolphin liver and human serum. The PFPeA qualifier MRM transition ion (m/z = 219 > 69) has been successfully applied in all the matrices with spike recoveries (90–125%), signal to noise ratios (>10) and transition ions ratio variation (80–120%). The PFBA qualifier pseudo-MRM transition ion (m/z = 169 > 169) works well in all the matrices except dolphin liver sample. The interpretation of pseudo-MRM results should be with cautions due to lower specificity compared to MRM. In addition, this project indicated under typical chromatographic conditions the MRM isobaric interference does happen frequently to PFPeA quantifier transition ion (m/z = 263 > 219) in serum and fish composite samples, and to PFBA quantifier transition ion (m/z = 213 > 169) in macrophytes, fish composite and dolphin liver samples.

Microplastics and Per- and Polyfluoroalkyl Substances (PFAS) Analysis in Sea Turtles and Bottlenose Dolphins along Mississippi’s Coast

Global plastic production and usage has increased annually for decades and microplastic pollutants (≤5 mm) are a growing concern. Microplastics in surface waters can adsorb and desorb harmful chemicals such as per- and polyfluoroalkyl substances (PFAS). Microplastics can accumulate across all tropic levels in the marine food web. The purpose of this research was to analyze the stomach and intestinal contents of stranded (Mississippi coast) bottlenose dolphins and sea turtles for the presence of microplastics and commonly found PFAS, PFOS, PFOA, and GenX. Gut contents were digested (10% KOH in 50% MeOH) and then analyzed for microplastics using pyrolysis gas chromatography-mass spectrometry (Pyro-GC-MS), Nile red microscopy, X-ray photo electron spectroscopy (XPS), and Raman spectroscopy. Digested sample filtrate was pre-concentrated using solid-phase extraction (SPE) before PFAS liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The PFOS extraction and analysis had 98.6% recovery when validated with certified pike‒perch fish reference material. The Nile red testing on most samples revealed the presence of microplastics (Table S1). The Pyro-GC-MS results from two samples confirmed the presence of the plasticizer acetamide. The Raman spectroscopy analysis indicated characteristic plastic peaks corresponding to polystyrene in one sample. PFOS (95.5 to 1,934.5 µg/kg) was detected in three dolphin stomach samples. This project is part of a long-term study with the goal of a better understanding of microplastics and PFAS environmental contamination and their impact on bottlenose dolphins and sea turtles.

Long-Term Monitoring of Polybrominated Diphenyl Ethers (PBDEs) in Indo-Pacific Humpback Dolphins (Sousa chinensis) from the Pearl River Estuary Reveals High Risks for Calves due to Maternal Exposure.

Although many studies have documented the wide occurrence of polybrominated diphenyl ethers (PBDEs) in cetaceans, little evidence exists regarding the detrimental effects of PBDE exposure on calf death rates for free-ranging cetaceans. This study analyzed life-history-associated PBDE bioaccumulation patterns in 128 stranding Indo-Pacific humpback dolphin (Sousa chinensis) samples over an 18-year timespan from the Pearl River Estuary (PRE). In comparison to the records of PBDE levels in cetaceans worldwide, the median levels of PBDEs (median = 10600 ng g-1 lw, range = 721-50900 ng g-1 lw) in all samples were the highest to date. One-way analysis of variance (ANOVA) showed that adult males (median = 16100 ng g-1 lw, range = 4070-50900 ng g-1 lw) and calves (12000 ng g-1 lw, range = 1250-35300 ng g-1 lw) both had the highest levels of PBDEs compared to the rest of the age/sex groups (p < 0.05). Concentrations of PBDEs in noncalves significantly decreased over the studied period, while those in calves had a slightly increasing trend, which may be due to different exposure routes via fish or milk, respectively. A significant and positive relationship was found between annual calf stranding death rates and body-length-adjusted PBDE concentrations in calves (r = 0.62, p < 0.05), suggesting that maternal exposure of calves to elevated levels of PBDEs may have contributed to the high annual stranding death rates of calves in the last two decades.

Body burdens and distribution of mercury and selenium in bottlenose, striped and Risso's dolphins along the Adriatic coast: A 20-year retrospective

Top marine predators present high mercury concentrations in their tissues due to biomagnification in the marine food chain. This study reports mercury (Hg) and selenium (Se) status, and the Hg:Se molar ratio assessment in bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's dolphins (Grampus griseus). Total Hg and Se concentrations were determined in muscle, liver, kidney, lung, spleen, adipose tissue and skin collected from 186 specimens stranded in the Croatian part of Adriatic Sea from 1995 to 2014. Total Hg concentrations in tissue samples ranged from 0.001 in the spleen to 2238 mg/kg wet weight in liver. Se concentrations in dolphin samples ranged from 0.010 to 2916 mg/kg ww. Minimum Se concentration was found in muscle and maximum Se concentration were found in liver of bottlenose dolphin. Hg and Se levels in Risso's dolphins showed higher concentrations in all tissues in comparison to bottlenose and striped dolphins. Significant and positive correlations were observed between age and Hg concentrations (P < 0.05). In 66.6 % of Risso's, 15.3 % of bottlenose dolphins and one stranded striped dolphin in this study, the hepatic concentration of Hg exceeded the higher toxic thresholds (400 mg/kg w.w.) previously defined as evidence of liver damage in marine mammals. The Hg:Se molar ratio in the liver of Risso's dolphin was 0.670. The liver of adult bottlenose dolphins showed expected values (0.870), while the liver of young dolphins had a high ratio (0.750), non-specific for the age group. The Hg:Se molar ratio in the liver of striped dolphins was 0.390, which is lower than the literature values.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets of bottlenose dolphins (Tursiops truncatus).

Marine mammals may suffer alterations in platelet function and hemostasia due to multiple pathologies, environmental conditions (including stress) or exposure to different contaminants that induce platelet activation. Detecting early alterations in platelet function in these animals could be an especially relevant diagnostic tool in these species because they typically do not show signs of weakness or disease until the pathology is in advanced state, in order to avoid attracting predators in natural conditions. The study of early markers of platelet activation is relevant for the detection, monitoring and therapy of inflammation and hemostasis disorders. Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular Ca2+ , using sensitive fluorescent indicators that can be loaded into intact cells. In order to study intraplatelet Ca2+ mobilization in marine mammals, we have adapted a kinetic assay of human platelet activation to study platelet activation in whole-blood samples of bottlenose dolphins (Tursiops truncatus) using the Ca2+ -sensitive dye Fluo-4AM and a clone of the platelet-specific antibody CD41-PE that recognizes dolphin platelets. This no-wash, no-lyse protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to platelet agonists under near-physiological conditions. The adaptation of this technique to marine mammals represents a methodological advance for basic and clinical veterinary applications but also for general environmental studies on these species.

Long-term increase in mortality of Indo-Pacific humpback dolphins (Sousa chinensis) in the Pearl River Estuary following anthropic activities: Evidence from the stranded dolphin mortality analysis from 2003 to 2017

With the dramatic increase in anthropogenic threats to the Pearl River Estuary (PRE), the population size of the Indo-Pacific humpback dolphins (Sousa chinensis) has significantly decreased over the past decade. To understand the impact and potential risks of intense human activities on these dolphins, factors related to the mortality of humpback dolphins in the PRE were investigated by a detailed examination of 343 dolphin specimens stranded during 2003–2017. There was a significant (p < 0.01) increasing trend for humpback dolphin stranding, reflecting the accelerating rate of the population decline. A large proportion of strandings (35.88%) were neonates. A low recruitment rate implies slow population growth, and hence, limited capacity to resist anthropogenic stress. The most commonly diagnosed causes of death were vessel collision and net entanglement. The concentrations of trace metals, polychlorinated biphenyl (PCB) congeners, dichlorodiphenyltrichloroethane, polycyclic aromatic hydrocarbons, and most of per- and polyfluoroalkyl substances (PFASs) in the dolphin samples were greater than those previously reported in cetaceans globally. Furthermore, Cu, PCB77, PCB169, PCB81, PCB37, and PFASs (excluding PFBA, PFPeA, PFHxA, PFHxDA, and PFODA) were the major pollutants accumulated in neonates. 67% of PCB, 78% of Cu, and 100% of perfluorooctane sulfonate concentrations in the neonates exceeded the threshold for toxicological effects in marine mammals, suggesting that these compounds could be important factors contributing to the low survival rate of calves in this area. This study revealed that vessel transportation, fishing activities, and pollutant bioaccumulation are the three major causes of humpback dolphin mortality in the PRE. These results highlight the need for more efforts to restrict anthropogenic activities, especially vessel traffic, the catching of these marine animals and fishing, and pollutant discharge, in order to prevent vulnerable species from continuous population decline and further extinction.

Assessing common bottlenose dolphin (Tursiops truncatus) population structure in Mississippi Sound and coastal waters of the north central Gulf of Mexico

Abstract Population structure of highly mobile marine organisms can be complex and difficult to study, but it is important to understand how populations partition themselves within their environment for accurate assessment of both natural and anthropogenic impacts and successful management. The 2010 Deepwater Horizon oil spill negatively impacted common bottlenose dolphins (Tursiops truncatus) within Mississippi Sound and the surrounding north central Gulf of Mexico (GOMx); however, little was known about their underlying population structure in these waters. Thus, it was unclear how many demographically independent populations were affected by the spill. Common bottlenose dolphin samples were collected throughout inshore waters of Mississippi Sound and coastal waters of the north‐central GOMx. Mitochondrial DNA control region sequence data and 19 nuclear microsatellite loci were analysed to determine how many populations are present and characterize their range throughout these waters. Bayesian clustering and migration analyses identified two genetically distinct and demographically independent populations: one predominantly inhabiting Mississippi Sound and adjacent coastal waters, and a second population extending generally from offshore of Mobile Bay, Alabama, east along the Florida Panhandle. Neither of these populations align with the currently delineated management stocks previously used to estimate impacts from the oil spill on common bottlenose dolphins in this portion of the GOMx. These results suggest that revisions may be necessary so that management stocks accurately represent the demographically independent populations present in these waters. Furthermore, better comprehension of underlying population structure will enhance impact assessments on common bottlenose dolphins and provide more appropriate baseline data to support future restoration and conservation objectives.

Alkenones in oceanic odontocetes as a potential proxy of environmental water temperature

The alkenones C37:2 and C37:3 are produced exclusively by some haptophyte species. Their relative proportion (Uk’37 index) may be used to infer the water temperature where the synthesising haptophyte lived. Alkenones have been analysed in sediments, planktonic communities and in the low trophic level, filter-feeding fin whales. However, it is unclear whether they can be detected in animals exploiting high trophic levels and used to infer the water temperature in which they live. Alkenones were analysed in blubber samples from three Mediterranean predatory species: the striped dolphin, an epipelagic species; the Risso’s dolphin, a deep diver; and the bottlenose dolphin, a coastal species. Alkenones were detected in all striped dolphin samples and in most of the Risso’s dolphin samples, but they were below detection limits in the bottlenose dolphin samples. The inferred temperature for the striped dolphins (16.4 ± 3.3 °C) was similar to the average water temperature of the region (16.9 ± 3.9 °C), but that for the Risso’s dolphins was lower than expected (12.7 ± 4.4 °C). The small sample sizes and the large variance in the Uk’37 index make it difficult to ascertain if the dissimilarity between the two oceanic species is real. Although further research is needed to calibrate this bio-indicator, we can conclude that alkenones are transferred through the trophic web and are found in oceanic cetaceans situated at a high trophic level.

First Report of Paralytic Shellfish Toxins in Marine Invertebrates and Fish in Spain.

A paralytic shellfish poisoning (PSP) episode developed in summer 2018 in the Rías Baixas (Galicia, NW Spain). The outbreak was associated with an unprecedentedly intense and long-lasting harmful algal bloom (HAB) (~one month) caused by the dinoflagellate Alexandrium minutum. Paralytic shellfish toxins (PSTs) were analyzed in extracts of 45 A. minutum strains isolated from the bloom by high-performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-PCOX-FLD). PSTs were also evaluated in tissues from marine fauna (invertebrates and fish) collected during the episode and in dolphin samples. The analysis of 45 A. minutum strains revealed a toxic profile including GTX1, GTX2, GTX3 and GTX4 toxins. With regard to the marine fauna samples, the highest PSTs levels were quantified in bivalve mollusks, but the toxins were also found in mullets, mackerels, starfish, squids and ascidians. This study reveals the potential accumulation of PSTs in marine invertebrates other than shellfish that could act as vectors in the trophic chain or pose a risk for human consumption. To our knowledge, this is the first time that PSTs are reported in ascidians and starfish from Spain. Moreover, it is the first time that evidence of PSTs in squids is described in Europe.

Marine mammals are natural hosts of Oceanivirga salmonicida, a bacterial pathogen of Atlantic salmon.

During 1992 and 1993, a bacterial disease occurred in a seawater Atlantic salmon Salmo salar farm, causing serious mortalities. The causative agent was subsequently named as Oceanivirga salmonicida, a member of the Leptotrichiaceae. Searches of 16S rRNA gene sequence databases have shown sequence similarities between O. salmonicida and uncultured bacterial clones from the digestive tracts of marine mammals. In the current study, oral samples were taken from stranded dolphins (common dolphin Delphinus delphis, striped dolphin Stenella coeruleoalba) and healthy harbour seals Phoca vitulina. A bacterium with growth characteristics consistent with O. salmonicida was isolated from a common dolphin. The isolate was confirmed as O. salmonicida, by comparisons to the type strain, using 16S rRNA gene, gyrB, groEL, and recA sequence analyses, average nucleotide identity analysis, and MALDI-TOF mass spectrometry. Metagenomic analysis indicated that the genus Oceanivirga represented a significant component of the oral bacterial microbiomes of the dolphins and seals. However, sequences consistent with O. salmonicida were only found in the dolphin samples. Analyses of marine mammal microbiome studies in the NCBI databases showed sequences consistent with O. salmonicida from the common dolphin, striped dolphin, bottlenose dolphin Tursiops truncatus, humpback whale Megaptera novaeangliae, and harbour seal. Sequences from marine environmental studies in the NCBI databases showed no sequences consistent with O. salmonicida. The findings suggest that several species of marine mammals are natural hosts of O. salmonicida.

Community composition and antibiotic resistance of bacteria in bottlenose dolphins Tursiops truncatus – Potential impact of 2010 BP Oil Spill

Aquatic contamination, oil spills in particular, could lead to the accumulation of antibiotic resistance by promoting selection for and/or transfer of resistance genes. However, there have been few studies on antibiotic resistance in marine mammals in relation to environmental disturbances, specifically oil contaminations. Here we initiated a study on antibiotic resistance bacteria in bottlenose dolphins Tursiops truncatus in relation to oil contamination following the 2010 BP Oil Spill in the northern Gulf of Mexico. Bacterial communities and antibiotic resistance prevalence one year after the 2010 BP Oil Spill were compared between Barataria Bay (BB) and Sarasota Bay (SB) by applying the rarefaction curve method, and (generalized) linear mixed models. The results showed that the most common bacteria included Vibrio, Shewanella, Bacillus and Pseudomonas. The prevalence of antibiotic resistance was high in the bacterial isolates at both bays. Though bacterial diversity did not differ significantly among water or dolphin samples, and antibiotic resistance did not differ significantly among water samples between the two bays, antibiotic resistance and multi-drug resistance in dolphin samples was significantly higher in the BB than in the SB, mainly attributed to the resistance to E, CF, FEP and SXT. We also found sulfamethoxazole-trimethoprim-resistant Stenotrophomonas maltophilia the first time in the natural aquatic environment. The higher antibiotic resistance in the dolphins in BB is likely attributed to 2010 BP Oil Spill as we expected SB, a more urbanized bay area, would have had higher antibiotic resistance based on the previous studies. The antibiotic resistance data gathered in this research will fill in the important data gaps and contributes to the broader spatial-scale emerging studies on antibiotic resistance in aquatic environments.

Persistent Organic Pollutants and Mercury in Genetically Identified Inner Estuary Bottlenose Dolphin (Tursiops truncatus) Residents of the Guayaquil Gulf, Ecuador: Ecotoxicological Science in Support of Pollutant Management and Cetacean Conservation

The bottlenose dolphin is the most common cetacean found in the coastal waters, estuaries and mangroves of Ecuador. However, its population size is gradually declining in the Gulf of Guayaquil, and anthropogenic factors including habitat degradation, uncontrolled dolphin watching, dredging activities, increasing maritime traffic, underwater noise, bycatch and marine pollution have been implicated in the decline. Very little is known concerning contamination by persistent organic pollutants (POPs) and mercury in bottlenose dolphins from the Pacific coast of South America. To address this research gap, the first assessment of total mercury (THg) and POPs, including polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and polybrominated diphenyl ethers (PBDEs), in free-ranging bottlenose dolphins in mangroves (El Morro Mangrove Wildlife Refuge) of the Gulf of Guayaquil, was conducted in Ecuador in 2018. Dolphin samples (i.e. skin and blubber; n = 9), were obtained using dart biopsy field methods for contaminant analysis. POP concentrations ranged 0.60-16.4 mg/kg lipid weight, while THg ranged from 1.40 to 4.0 mg/kg dry weight. The predominant POPs were OCPs (51% of ∑POP), followed by PCBs (44%) and PBDEs (5.5%); particularly, p,p’-DDE, the main DDT metabolite and a potent anti-androgenic, accounted by 44% of ∑POP, ranging 0.12 to~9.0 mg/kg lw, followed by PCB 153 (8.0%) and PCB 180 (5.0%). PBDE 47 accounted for 2.0% of ∑POP. While the POP concentrations are lower than those found in dolphins from many other regions of the world, some of the THg concentrations are within the concentration range found in dolphins from the southeastern coast of the USA. The ecotoxicological risk assessment showed that some of the sampled dolphin are exposed to immunotoxic and endocrine disruption effects by POPs and mercury. The low genetic diversity of this distinctive dolphin population, likely exhibiting genetic isolation and a unique evolutionary heritage, could be lost if the population continues to decline in the face of anthropogenic threats, including chemical pollution. Our finding shows that bottlenose dolphins in coastal Ecuador are exposed to environmental contaminants and can be used as sentinel species of ecosystem health to monitor pollution in the region to support ecotoxicological risk assessment and regional pollutant management.

1H NMR metabolomic analysis of skin and blubber of bottlenose dolphins reveals a functional metabolic dichotomy

The common bottlenose dolphin (Tursiops truncatus) is a carnivorous cetacean that thrives in marine environments, one of the apex predators of the marine food web. They are found in coastal and estuarine ecosystems, which are known to be sensitive to environmental impacts. Dolphins are considered sentinel organisms for monitoring the health of coastal marine ecosystems due to their role as predators that can bioaccumulate contaminants. Although recent studies have focused on capturing the circulating metabolomes of these mammals, and in the context of pollutants and exposures in the marine environment, skin and blubber are important surface and protective tissues that have not been adequately probed for metabolism. Using a proton nuclear magnetic resonance spectroscopy (1H NMR) based metabolomics approach, we quantified 51 metabolites belonging to 74 different metabolic pathways in the skin and blubber of stranded bottlenose dolphin (n = 4) samples collected at different localities in the Southern Zone coast of Yucatan Peninsula of Mexico. Results indicate that metabolism of skin and blubber are quantitatively very different. These metabolite abundances could help discriminate the tissue-types using supervised partial least square regression discriminant analysis (PLSDA). Further, using hierarchical clustering analysis and random forest analysis of the metabolite abundances, the results pointed to unique metabolites that are important classifiers of the tissue-type. On one hand, the differential metabolic patterns, mainly linking fatty acid metabolism and ketogenic amino acids, seem to constitute a characteristic of blubber, thus pointing to fat synthesis and deposition. On the other hand, the skin showed several metabolites involved in gluconeogenic pathways, pointing towards an active anabolic energy-generating metabolism. The most notable pathways found in both tissues included: urea cycle, nucleotide metabolism, amino acid metabolism, glutathione metabolism among others. Our 1H NMR metabolomics analysis allowed the quantification of metabolites associated with these two organs, i.e., pyruvic acid, arginine, ornithine, 2-hydroxybutyric acid, 3-hydroxyisobutyric acid, and acetic acid, as discriminatory and classifying metabolites. These results would lead to further understanding of the functional and physiological roles of dolphin skin and blubber metabolism for better efforts in their conservation, as well as useful target biopsy tissues for monitoring of dolphin health conditions in marine pollution and ecotoxicology studies.