Survey of severe acute respiratory syndrome coronavirus 2 in captive and free-ranging wildlife from Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), considered a zoonotic agent of wildlife origin, can infect various animal species, including wildlife in free-range and captive environments. Detecting susceptible species and potential reservoirs is crucial for preventing the transmission, spread, genetic evolution, and further emergence of viral variants that are major threats to global health. This study aimed to detect exposure or acute infection by SARS-CoV-2 in 420 animals from 40 different wildlife species, including terrestrial and aquatic mammals, from different regions of Spain during the 2020-2023 coronavirus disease 19 (COVID-19) pandemic. In total, 8/137 animals were positive for SARS-CoV-2 antibodies against the receptor binding domain and/or viral nucleoprotein according to independent ELISAs. However, only one ELISA-positive sample of a captive bottlenose dolphin (Tursiops truncatus) tested positive for SARS-CoV-2 neutralizing antibodies with a low titre (SNT50 38.15) according to a virus neutralization test. Cetaceans are expected to have a high risk of infection with SARS-CoV-2 according to early predictive studies due to the similarity of their angiotensin converting enzyme 2 cell receptor to that of humans. Moreover, of 283 animals analysed for SARS-CoV-2 RNA using RT-qPCR, none tested positive. Our results reinforce the importance of considering cetaceans at risk for SARS-CoV-2 infection and support taking preventive biosecurity measures when interacting with them, especially in the presence of individuals with suspected or confirmed COVID-19. Although most animals in this study tested negative for acute infection or viral exposure, ongoing surveillance of wildlife species and potentially susceptible animals is important to prevent future spillover events and detect potential novel reservoirs.

Evaluation of plasma human atrial natriuretic peptide concentration in healthy bottlenose dolphins (Tursiops truncates).

There are currently no standard methods for diagnosing cardiac diseases in dolphins. These diseases may consequently be overlooked and go undiagnosed. The presence and severity of cardiac diseases in humans can be determined using blood tests. Human atrial natriuretic peptide (hANP) used in human cardiac examinations has low species specificity. There have already been reports of homology between dolphin ANP and hANP; however, its potential for clinical application in dolphins has not been tested. This study was conducted to establish a reference for plasma hANP levels in healthy bottlenose dolphins. Healthy bottlenose dolphins (seven females; estimated to be 7-30 years of age) at an aquarium in Japan were sampled. Each animal was tested for hANP at least three times, and the mean value and standard deviation were calculated to be 43.4 ± 19.2 pg/mL. In humans, patients with high plasma hANP levels have a poor prognosis. In veterinary medicine, cutoff values for the diagnosis of mitral regurgitation and heart failure in dogs have been established and used to predict prognosis. The results of the present study may contribute to the health management of bottlenose dolphins, particularly in the early detection and treatment of cardiac disease.

Effects of a simple method for determining the time of insemination and different methods on artificial insemination in common bottlenose dolphins (Tursiops truncatus).

In this study, we examined the usefulness of a simpler and more feasible method for determining the optimal timing of artificial insemination and the conditions for its success in six female common bottlenose dolphins. Pregnancy was successfully achieved in five dolphins by performing intrauterine insemination, using chilled semen stored for less than 3 days or frozen semen within 24 hr of exhibiting a peak serum estradiol (E2) level of 100 pg/mL or higher or on the day with a serum E2 level of approximately 100 pg/mL, measured with a simple measuring device. We concluded that the determining the optimal timing of intrauterine insemination by measuring serum E2 levels is a simpler and more useful method compared with the conventional approach.

Juvenile social play predicts adult reproductive success in male bottlenose dolphins.

For over a century, the evolution of animal play has sparked scientific curiosity. The prevalence of social play in juvenile mammals suggests that play is a beneficial behavior, potentially contributing to individual fitness. Yet evidence from wild animals supporting the long-hypothesized link between juvenile social play, adult behavior, and fitness remains limited. In Western Australia, adult male bottlenose dolphins (Tursiops aduncus) form multilevel alliances that are crucial for their reproductive success. A key adult mating behavior involves allied males using joint action to herd individual females. Juveniles of both sexes invest significant time in play that resembles adult herding-taking turns in mature male (actor) and female (receiver) roles. Using a 32-y dataset of individual-level association patterns, paternity success, and behavioral observations, we show that juvenile males with stronger social bonds are significantly more likely to engage in joint action when play-herding in actor roles. Juvenile males also monopolized the actor role and produced an adult male herding vocalization ("pops") when playing with females. Notably, males who spent more time playing in the actor role as juveniles achieved more paternities as adults. These findings not only reveal that play behavior provides male dolphins with mating skill practice years before they sexually mature but also demonstrate in a wild animal population that juvenile social play predicts adult reproductive success.

Platelet phosphatidylserine exposure and microparticle production as health bioindicators in marine mammals.

In human medicine, various pathologies, including decompression sickness, thrombocytopenia, and rheumatoid arthritis, have been linked to changes in cellular microparticles (MP) formation, particularly platelet microparticles (PMP). Similar disorders in marine mammals might be attributed to anthropogenic threats or illnesses, potentially impacting blood PMP levels. Thus, detecting platelet phosphatidylserine (PS) exposure and PMP formation could serve as a crucial diagnostic and monitoring approach for these conditions in marine mammals. Our group has developed a methodology to assess real-time PS exposure and PMP formation specifically tailored for marine mammals. This method, pioneered in species such as bottlenose dolphins, beluga whales, walruses, and California sea lions, represents a novel approach with significant implications for both clinical assessment and further research into platelet function in these animals. The adapted methodology for evaluating PS exposure and PMP formation in marine mammals has yielded promising results. By applying this approach, we have observed significant correlations between alterations in PMP levels and specific pathologies or environmental factors. These findings underscore the potential of platelet function assessment as a diagnostic and monitoring tool in marine mammal health. The successful adaptation and application of this methodology in marine mammals highlight its utility for understanding and managing health concerns in these animals.

In pop pursuit: social bond strength predicts vocal synchrony during cooperative mate guarding in bottlenose dolphins.

Vocal communication is an emblematic feature of group-living animals, used to share information and strengthen social bonds. Vocalizations are also used to coordinate group-level behaviours in many taxa, but little is known of the factors that may influence vocal behaviour during cooperative acts. Allied male Indo-Pacific bottlenose dolphins (Tursiops aduncus) use the 'pop' vocalization as a coercive signal when working together to herd single oestrous females. Using long-term association and acoustic data, we examined the influence of social and non-social factors on pop use by allied male dolphins in this context. Neither pop rate nor pop bout duration were influenced by any of the factors examined. However, allied males with stronger social bonds engaged in higher rates of vocal synchrony; whereby they actively matched the timing of their pop production. Hence, social bond strength influenced pop use in a cooperative context, suggesting dual functions of pop use: to induce the female to remain close, and to promote social bond maintenance and cooperation among males. This article is part of the theme issue 'The power of sound: unravelling how acoustic communication shapes group dynamics'.

Spatial Variation in Mercury Accumulation in Bottlenose Dolphins (Tursiops spp.) in Southeastern U.S.A.

Bottlenose dolphins (Tursiops spp.) inhabit bays, sounds, and estuaries (BSEs) throughout the southeast region of the U.S.A. and are sentinel species for human and ecosystem-level health. Dolphins are vulnerable to the bioaccumulation of contaminants through the coastal food chain because they are high-level predators. Currently, there is limited information on the spatial dynamics of mercury accumulation in these dolphins. Total mercury (THg) was measured in dolphin skin from multiple populations across the U.S. Southeast Atlantic and Gulf of Mexico coasts, and the influence of geographic origin, sex, and age class was investigated. Mercury varied significantly among sampling sites and was greatest in dolphins in St. Joseph Bay, Florida Everglades, and Choctawhatchee Bay (14,193 ng/g ± 2196 ng/g, 10,916 ng/g ± 1532 ng/g, and 7333 ng/g ± 1405 ng/g wet mass (wm), respectively) and lowest in dolphins in Charleston and Skidaway River Estuary (509 ng/g ± 32.1 ng/g and 530 ng/g ± 58.4 ng/g wm, respectively). Spatial mercury patterns were consistent regardless of sex or age class. Bottlenose dolphin mercury exposure can effectively represent regional trends and reflect large-scale atmospheric mercury input and local biogeochemical processes. As a sentinel species, the bottlenose dolphin data presented here can direct future studies to evaluate mercury exposure to human residents in St. Joseph Bay, Choctawhatchee Bay, and Florida Coastal Everglades, as well as additional sites with similar geographical, oceanographic, or anthropogenic parameters. These data may also inform state and federal authorities that establish fish consumption advisories to determine if residents in these locales are at heightened risk for mercury toxicity.

An update on the development of a bottlenose dolphin, Tursiops truncatus, immune reagent toolkit.

There are extensive immunological reagents available for laboratory rodents and humans. However, for veterinary species there is a need for expansion of immunological toolkits, with this especially evident for marine mammals, such as cetaceans. In addition to their use in a research setting, immune assays could be employed to monitor the health status of cetaceans and serve as an adjunct to available diagnostic tests. Such development of specific and sensitive immune assays will enhance the proper care and stewardship of wild and managed cetacean populations. Our goal is to provide immune reagents and immune assays for the research community, clinicians, and others involved in care of bottlenose dolphins. This review will provide an update on our development of a bottlenose dolphin immunological toolkit. The future availability and continued development of these reagents is critical for improving wild and managed bottlenose dolphin population health through enhanced assessment of their responses to alterations in the marine environment, including pathogens, and improve our ability to monitor their status following vaccination.

Investigation of the Link between Per- and Polyfluoroalkyl Substances and Stress Biomarkers in Bottlenose Dolphins (Tursiops truncatus).

Bottlenose dolphins (Tursiops truncatus) are keystone and sentinel species in the world's oceans. We studied correlations between per- and polyfluoroalkyl substances (PFAS) and their stress axis. We investigated associations between plasma biomarkers of 12 different PFAS variants and three cortisol pools (total, bound, and free) in wild T. truncatus from estuarine waters of Charleston, South Carolina (n = 115) and Indian River Lagoon, Florida (n = 178) from 2003 to 2006, 2010-2013, and 2015. All PFAS and total cortisol levels for these dolphins were previously reported; bound cortisol levels and free cortisol calculations have not been previously reported. We tested null hypotheses that levels of each PFAS were not correlated with those of each cortisol pool. Free cortisol levels were lower when PFOS, PFOA, and PFHxS biomarker levels were higher, but free cortisol levels were higher when PFTriA was higher. Bound cortisol levels were higher when there were higher PFDA, PFDoDA, PFDS, PFTeA, and PFUnDA biomarkers. Total cortisol was higher when PFOA was lower, but total cortisol was higher when PFDA, PFDoDA, PFTeA, and PFTriA were higher. Additional analyses indicated sex and age trends, as well as heterogeneity of effects from the covariates carbon chain length and PFAS class. Although this is a cross-sectional observational study and, therefore, could reflect cortisol impacts on PFAS toxicokinetics, these correlations are suggestive that PFAS impacts the stress axis in T. truncatus. However, if PFAS do impact the stress axis of dolphins, it is specific to the chemical structure, and could affect the individual pools of cortisol differently. It is critical to conduct long-term studies on these dolphins and to compare them to populations that have no or little expose to PFAS.

Apneustic anesthesia ventilation improves pulmonary function in anesthetized bottlenose dolphins (Tursiops truncatus).

Introduction: Use of mechanical ventilation during general anesthesia is a necessary practice in the anesthetization of small cetaceans as spontaneous ventilation fails to provide adequate gas exchange. Currently available methods of ventilation do not account for the intermittent breathing strategy of representative species within this infraorder of fully aquatic mammals and may have a significant effect on cardiac and respiratory physiology. Methods: To understand the impact of mechanical ventilation on cardiopulmonary function in one small species of cetacean, the bottlenose dolphin (Tursiops truncatus), we compared controlled mechanical ventilation (CMV) to a novel ventilation method known as apneustic anesthesia ventilation (AAV). AAV simulates the normal inspiratory breath-hold pattern of dolphins. Ten anesthetic procedures (dental procedure, n = 9; bronchoscopy, n = 2) were performed on nine dolphins (age range: 10-42 years; mean = 32 years; median = 37 years; female = 3, 40%; male = 6, 60%). In a cross-over study design, dolphins were instrumented and randomly assigned to AAV or CMV as the initial mode of ventilation, then switched to the alternate mode. Baseline cardiopulmonary data were collected and again after 30 min on each mode of ventilation. Cardiac index, stroke volume index, systemic vascular resistance, alveolar dead space, alveolar-arterial oxygen tension gradient, arterial oxygen content, oxygen delivery index, and dynamic respiratory system compliance index were calculated at each of the four time points. Results: During AAV, dolphins had higher arterial oxygen tension, higher mean airway pressure, reduced alveolar dead space ventilation and lower alveolar-arterial oxygen difference. Cardiovascular performance was not statistically different between the two modes. Discussion: Our study suggests AAV, which more closely resembles the conscious intermittent respiratory pattern phenotype of dolphins, improves ventilation and pulmonary function in the anesthetized dolphin. Future studies should evaluate the cardiopulmonary effects of neutral buoyancy and cardiopulmonary sparing drug protocols to reduce the need for hemodynamic support of current protocols.

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

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.

First description of Rhodococcus equi infection in common bottlenose dolphin (Tursiops truncatus).

Rhodococcus equi is a terrestrial bacterium and a common pathogen in foals (Equus caballus), in which causes pneumonia. This report describes for the first time the infection caused by R. equi in a common bottlenose dolphin (Tursiops truncatus) stranded in the Calabrian coast, Italy. The post mortem examination of the animal revealed lesions in lung and colon. The animal was also positive to dolphin morbillivirus. The histological study showed lesions attributable to R. equi infection, such as pyogranulomatous bacterial pneumonia and chronic granulomatous colitis. Whole genome sequencing of the isolated strain confirmed its identification as R. equi.

Distribution of calcium-binding proteins immunoreactivity in the bottlenose dolphin entorhinal cortex.

Introduction: The entorhinal cortex has been shown to be involved in high-level cognitive functions in terrestrial mammals. It can be divided into two main areas: the lateral entorhinal area (LEA) and the medial entorhinal area (MEA). Understanding of its structural organization in cetaceans is particularly important given the extensive evidence for their cognitive abilities. The present study describes the cytoarchitectural and immunohistochemical properties of the entorhinal cortex of the bottlenose dolphin (Tursiops truncatus, Montagu, 1821), perhaps the most studied cetacean species and a paradigm for dolphins and other small cetaceans. Methods: Four bottlenose dolphins' entorhinal cortices were processed. To obtain a precise overview of the organization of the entorhinal cortex we used thionin staining to study its laminar and regional organization, and immunoperoxidase technique to investigate the immunohistochemical distribution of three most commonly used calcium-binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV). Entorhinal cortex layers thickness were measured, morphological and morphometric analysis for each layer were conducted and statistically compared. Results: Six layers in both the LEA and MEA were identified. The main difference between the LEA and the MEA is observed in layers II and III: the neurons in layer II of the LEA were denser and larger than the neurons in layer II of MEA. In addition, a relatively cell-free zone between layers II and III in LEA, but not in MEA, was observed. The immunohistochemical distribution of the three CBPs, CB, CR and PV were distinct in each layer. The immunostaining pattern of CR, on one side, and CB/PV, on the other side, appeared to be distributed in a complementary manner. PV and CB immunostaining was particularly evident in layers II and III, whereas CR immunoreactive neurons were distributed throughout all layers, especially in layers V and VI. Immunoreactivity was expressed by neurons belonging to different morphological classes: All CBPs were expressed in non-pyramidal neurons, but CB and CR were also found in pyramidal neurons. Discussion: The morphological characteristics of pyramidal and non-pyramidal neurons in the dolphin entorhinal cortex are similar to those described in the entorhinal cortex of other species, including primates and rodents. Interestingly, in primates, rodents, and dolphins, most of the CBP-containing neurons are found in the superficial layers, but the large CR-ir neurons are also abundant in the deep layers. Layers II and III of the entorhinal cortex contain neurons that give rise to the perforant pathway, which conveys most of the cortical information to the hippocampal formation. From the hippocampal formation, reciprocal projections are directed back to the deep layer of the entorhinal cortex, which distributes the information to the neocortex and subcortical area. Our data reveal that in the dolphin entorhinal cortex, the three major CBPs label morphologically heterogeneous groups of neurons that may be involved in the information flow between entorhinal input and output pathways.

Vascularization of the gastrointestinal tract of the bottlenose dolphin (Tursiops truncatus, Montagu 1821).

Odontocetes primarily rely on fish, cephalopods, and crustaceans as their main source of nutrition. In the digestive system, their polygastric complex exhibits similarities to that of their closest terrestrial relatives such as cows, sheep, and giraffes, while the entero-colic tract shares similarities with terrestrial carnivores. The morphology, caliber, and structure of the odontocete intestine are relatively constant, and, since there is no caecum, a distinction between the small and large intestine and their respective subdivisions is difficult. To address this issue, we used the intestinal vascularization pattern, specifically the course and branching of the celiac artery (CA) and the cranial and caudal mesenteric arteries (CrMA and CdMA). A series of pictures and dissections of 10 bottlenose dolphins (Tursiops truncatus) were analyzed. Additionally, we performed a cast by injecting colored polyurethane foam in both arteries and veins to measure the caliber of the arteries and clarify their monopodial or dichotomous branching. Our results showed the presence of multiple duodenal arteries (DAs) detaching from the CA. The CrMA gave origin to multiple jejunal arteries, an ileocolic artery (ICA), and, in six cases, a CdMA. In four specimens, the CdMA directly originated from the abdominal aorta. The ICA gave rise to the mesenteric ileal branches (MIB) and mesenteric anti-ileal branches and the right colic arteries (RCA) and the middle colic arteries. From the CdMA originated the left colic and cranial rectal arteries (LCA and CrRA). The measurements revealed a mixed monopodial and dichotomous branching scheme. The analysis of the arteries and their branching gave us an instrument, based on comparative anatomy, to distinguish between the different intestinal compartments. We used the midpoint of anastomoses between MIB and RCA to indicate the border between the small and the large intestine, and the midpoint of anastomoses between LCA and CrRA, to tell the colon from the rectum. This pattern suggested an elongation of the duodenum and a shortening of the colic tract that is still present in this species. These findings might be related to the crucial need to possess a long duodenal tract to digest prey ingested whole without chewing. A short aboral part is also functional to avoid gas-producing colic fermentation. The rare origin of the CdMA on the CrMA might instead be a consequence of the cranial thrust of the abdominopelvic organs related to the loss of the pelvic girdle that occurred during the evolution of cetaceans.

Optimization and validation of a micro-QuEChERS method for phthalates detection in small samples of cetacean blubber.

In this study, an innovative method was developed to detect and quantify phthalates in fresh cetacean blubber. An adaptation of the ammonium formate QuEChERS method was used and adapted as a micro-extraction for small quantities of samples. Significantly, this technique utilized minimal quantities of reagents and salts, with the additional implementation of rigorous Quality Assurance/Quality Control protocols to further reduce background contamination. To ensure the reliability of this method, comprehensive validation procedures were conducted, with a specific focus on two widely studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus). Determination coefficients (R2) for matrix-matched calibration were >0.93 with limits of quantifications (LOQ) of the method in the range of 5-10 ng/g. Mean recovery values were between 40 and 100 %. This novel methodology holds particular relevance for environmental research studies, offering the capability to detect emerging contaminants with minimal sample requirements. This aspect is particularly valuable in investigations that involve free-ranging animals and rely on biopsy sampling. It allows for the assessment of contaminant levels in healthy individuals within wild populations, enhancing our understanding of ecological impacts and potential conservation measures.•A micro-extraction adaptation of the ammonium formate QuEChERS method was developed and applied to a small quantity of fresh cetacean blubber to detect phthalates.•Small quantities of reagents and salts were used, and additional Quality Assurance/ Quality Control procedures were taken to further minimize background contamination.•Method validation was carried out for two cosmopolitan and extensively studied cetacean species: the common bottlenose dolphin (Tursiops truncatus) and the short-finned pilot whale (Globicephala macrorhynchus).

Electric boat underwater radiated noise and its potential impact on species of conservation interest.

Electric boats are thought to be noiseless, but in-situ measurements are generally rare. The Underwater Radiated Noise (URN) of 8-m Trimaran Pontoon Boat with two outboard electric engines was measured in the Miramare Marine Protected Area (Trieste, Italy), together with the URN of a fibreglass 5-m boat, with a outboard gasoline engine, for comparisons. International standards and guidelines for shallow waters were considered. URN were provided in one-third octave band and in narrow band spectra. The electric boat produced a low input of underwater noise at low frequencies. Given a low-frequency hearing sensitivity, the listening space reduction (LSR) was lower when generated by the electric than by combustion engine boat for the brown meagre, a local Teleost fish. No difference was found for the bottlenose dolphin LSR although continuous, tonal, high frequency components generated by the electric boat are expected to be highly detrimental for the bottlenose dolphin.

Establishment and characterization of a novel lung cell line derived from the common bottlenose dolphin.

Cetaceans are specialized marine mammals with a unique respiratory system adapted for diving behavior. Furthermore, respiratory diseases are commonly observed in these mammals. Nevertheless, much of their respiratory physiology remains unknown due to the limited supply and poor quality of their biological samples for research. In this study, we established a novel lung cell line, dLu, derived from the common bottlenose dolphin (Tursiops truncatus), which can prove useful in cetacean research, including for understanding the pathogenesis of respiratory diseases in cetaceans. The cells were cultured in a simple medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The morphology of the cells was fibroblast-like. dLu was produced by transfecting the simian virus 40 large T antigen into primary cultured cells. Although dLu exhibited approximately 80 cell divisions, it was unable to achieve complete immortalization, as the cells stopped proliferating beyond this number. dLu cells expressed toll-like receptor 3 but not toll-like receptor 4. Immunostimulation with poly(I:C) altered the gene expressions of interferon beta 1 and tumor necrosis factor alpha in dLu cells. In summary, dLu established in this study is a novel cetacean cell resource that can be easily cultured and is a useful in vitro tool in cetacean research, particularly for studying host immune responses in the lungs.

Vessels Disturb Bottlenose Dolphin Behavior and Movement in an Active Ship Channel.

Although the Port of Corpus Christi, Texas, has become a top oil exporter, it is unknown if local dolphins are disturbed by high year-round vessel traffic. A shore-based digital theodolite and automatic identification system receiver were used to record data to assess common bottlenose dolphin (Tursiops truncatus) behavioral states and movement patterns in the Corpus Christi Ship Channel (CCSC) in relation to vessel traffic. Multinomial logistic regression and generalized additive models were applied to analyze the data. Vessels were present within 300 m of dolphins during 80% of dolphin observations. Dolphins frequently foraged (40%), traveled (24%), socialized (15%), and milled (14%), but rarely oriented against the current (7%) or rested (1% of observations). Season, time of day, group size, vessel type, vessel size, and number of vessels were significant predictors of dolphin behavioral state. Significant predictors of dolphin movement patterns included season, time of day, group size, calf presence, vessel type, and vessel numbers. The CCSC is an important foraging area for dolphins, yet the high level of industrial activity puts the dolphins at risk of human-related disturbance and injury. There is a crucial need to monitor the impact of increased anthropogenic influences on federally protected dolphins in the active CCSC, with broad application to dolphins in other ports.

Isolation and Characterization of Cetacean Cell-Derived Extracellular Vesicles.

Cetaceans are of scientific interest because they are good candidates as environmental bioindicators. However, in vivo research is arduous and in vitro studies represent a rarely used valid alternative. Extracellular vesicles (EVs) are membrane-bound structures playing roles in cell-to-cell communication. Despite being a promising investigative tool in different fields of science, EVs have been poorly studied in cetaceans. To fill this gap, we describe the preliminary characterization of EVs isolated from a bottlenose dolphin and a Cuvier's beaked whale cell line. EVs have been isolated with ultracentrifugation (UC) or size exclusion chromatography (SEC) and characterized with nanoparticle tracking analysis (NTA), Western blotting (WB), and scanning transmission electron microscopy (STEM). UC and SEC allowed the isolation of mainly small EVs (<200 nm). A higher number of particles were isolated through UC compared to SEC from both cell lines. At WB, all EVs expressed the EV-markers CD9 and integrin-ß. Only EVs isolated with UC were positive for TSG101. In conclusion, we isolated for the first time EVs from a bottlenose dolphin and a Cuvier's beaked whale cell line using two different techniques. Further studies on cell-derived EVs will be useful to deepen our knowledge on cetacean pathophysiology and health status assessment.

Determination of water balance maintenance in Orcinus orca and Tursiops truncatus using oxygen isotopes.

The secondary adaptation of Cetacea to a fully marine lifestyle raises the question of their ability to maintain their water balance in a hyperosmotic environment. Cetacea have access to four potential sources of water: surrounding salt oceanic water, dietary free water, metabolic water and inhaled water vapour to a lesser degree. Here, we measured the 18O/16O oxygen isotope ratio of blood plasma from 13 specimens belonging to two species of Cetacea raised under human care (four killer whales Orcinus orca, nine common bottlenose dolphins Tursiops truncatus) to investigate and quantify the contribution of preformed water (dietary free water, surrounding salt oceanic water) and metabolic water to Cetacea body water using a box-modelling approach. The oxygen isotope composition of Cetacea blood plasma indicates that dietary free water and metabolic water contribute to more than 90% of the total water input in weight for cetaceans, with the remaining 10% consisting of inhaled water vapour and surrounding water accidentally ingested or absorbed through the skin. Moreover, the contribution of metabolic water appears to be more important in organisms with a more lipid-rich diet. Beyond these physiological and conservation biology implications, this study opens up questions that need to be addressed, such as the applicability of the oxygen isotope composition of cetacean body fluids and skeletal elements as an environmental proxy of the oxygen isotope composition of present and past marine waters.