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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.

AIMS: Gastrointestinal disease is a leading cause of morbidity in bottlenose dolphins (Tursiops truncatus) under managed care. Fecal microbiota transplantation (FMT) holds promise as a therapeutic tool to restore gut microbiota without antibiotic use. This prospective clinical study aimed to develop a screening protocol for FMT donors to ensure safety, determine an effective FMT administration protocol for managed dolphins, and evaluate the efficacy of FMTs in four recipient dolphins. METHODS AND RESULTS: Comprehensive health monitoring was performed on donor and recipient dolphins. Fecal samples were collected before, during, and after FMT therapy. Screening of donor and recipient fecal samples was accomplished by in-house and reference lab diagnostic tests. Shotgun metagenomics was used for sequencing. Following FMT treatment, all four recipient communities experienced engraftment of novel microbial species from donor communities. Engraftment coincided with resolution of clinical signs and a sustained increase in alpha diversity. CONCLUSION: The donor screening protocol proved to be safe in this study and no adverse effects were observed in four recipient dolphins. Treatment coincided with improvement in clinical signs.

"Gaze following"-when one individual witnesses another shift its orientation, and then re-orients in the same direction-has been observed in a wide range of species. Related work with dolphins has to date focused on human-dolphin interactions. In this conspecific study, we examined a group of dolphins orienting, in passing, to gateways between their pools, as opportunities for witnesses to demonstrate "gaze following". Seven bottlenose dolphins were synchronously videotaped on six underwater cameras, for 21 h over three days, and the recordings analyzed by trained observers. The identities of all animals present, their partner state, and whether and to what degree they had altered their access to the gate (e.g., from Monocular to Binocular, or Binocular to Visio-Echoic) was recorded. Compared to animals that did not witness such a change, witnesses of an increase in access by another dolphin were significantly more likely to also act to increase their own access. We observed 460 such cases of "gaze following" in these animals. Dolphins who were partnered (showed sustained swimming within 1 body length) were significantly more likely, than non-partnered animals, to "gaze follow". Dolphins also showed a significant tendency toward matching the kind of access they observed. No significant difference was found in the presence of animals in the back pools, during changes in orientation that were followed, versus in those that were not. These findings support adding bottlenose dolphins to the growing list of species that display conspecific "gaze following".

High-resolution non-invasive cetacean tagging systems can be used to investigate the influence of habitat characteristics and management factors on behavior by quantifying activity levels and distance traveled by bottlenose dolphins (Tursiops truncatus and Tursiops aduncus) in accredited zoos and aquariums. Movement Tags (MTags), a bio-logging device, were used to record a suite of kinematic and environmental information outside of formal training sessions as part of a larger study titled "Towards understanding the welfare of cetaceans in zoos and aquariums" (colloquially called the Cetacean Welfare Study). The purpose of the present study was to explore if and how habitat characteristics, environmental enrichment programs, and training programs were related to the distance traveled and energy expenditure of dolphins in accredited zoos and aquariums. Bottlenose dolphins in accredited zoos and aquariums wore MTags one day per week for two five-week data collection periods. Overall dynamic body acceleration (ODBA), a proxy for energy expenditure, and average distance traveled per hour (ADT) of 60 dolphins in 31 habitats were examined in relation to demographic, habitat, and management factors. Participating facilities were accredited by the Alliance for Marine Mammal Parks and/or Aquariums and the Association of Zoos & Aquariums. Two factors were found to be related to ADT while six factors were associated with ODBA. The results showed that enrichment programs were strongly related to both ODBA and ADT. Scheduling predictable training session times was also positively associated with ADT. The findings suggested that habitat characteristics had a relatively weak association with ODBA and were not related to ADT. In combination, the results suggested that management practices were more strongly related to activity levels than habitat characteristics.

Accredited zoos and aquariums continually strive to ensure high levels of animal welfare for the animals under their professional care. Best management practices include conducting research to better understand factors that lead to optimal welfare and then turning findings into practice. The current study is part of the larger Cetacean Welfare Study or more formally, "Towards understanding the welfare of cetaceans in zoos and aquariums." Facilities participating in the study were accredited by the Alliance of Marine Mammal Parks and Aquariums and/or the Association of Zoos and Aquariums. Animal management factors and habitat characteristics were examined in relation to two potential indicators of welfare for common (Tursiops truncatus) and Indo-Pacific (Tursiops aduncus) bottlenose dolphins. Specifically, we examined environmental enrichment, animal training, and habitat characteristics that were significantly related to behavioral diversity and route tracing, a form of stereotypic behavior. Behavior was recorded from 47 animals at 25 facilities around the world. Overall, the rate of route tracing behavior observed during the study was very low and few animal management factors or habitat characteristics were found to be related to this behavior. One factor, enrichment diversity, had a significant positive relationship with route tracing and an inverse relationship with behavioral diversity. This finding may be a product of a response mounted by animal care specialists to the behavior as opposed to a cause. Animals that engaged in this behavior were likely provided more diverse enrichment in attempts to ameliorate the stereotypic behavior. However, multiple factors were found to significantly relate to behavioral diversity, a potential positive indicator of welfare for bottlenose dolphins. Dolphins that were trained on a predictable schedule had higher behavioral diversity than those on a semi-predictable schedule. There was a positive significant relationship between behavioral diversity and the number of habitats to which an animal had access, and a significant inverse relationship with the maximum depth of the habitat. Finally, animals that were split into groups and reunited or rotated between subgroups had higher behavioral diversity than animals managed in the same group. Information gained from the current study suggested that animal management techniques may be more important in ensuring good welfare for bottlenose dolphins than focusing on habitat size.

The way an animal uses its habitat can serve as an indicator of habitat appropriateness for the species and individuals. Bottlenose dolphins (Tursiops truncatus and Tursiops aduncus) in accredited zoos and aquariums experience a range of habitat types and management programs that provide opportunities for dolphins to engage in species-appropriate behaviors and potentially influence their individual and group welfare. Data in the present study were collected as part of a larger study titled "Towards understanding the welfare of cetaceans in zoos and aquariums" (colloquially called the Cetacean Welfare Study). Non-invasive bio-logging devices (Movement Tags) recorded the diving behavior and vertical habitat movements of 60 bottlenose dolphins at 31 zoos and aquariums that were accredited by the Alliance for Marine Mammal Parks and Aquariums and/or the Association of Zoos & Aquariums. Bottlenose dolphins wore a Movement Tag one day per week for two five-week data collection periods. Demographic variables, environmental enrichment programs, training programs, and habitat characteristics were associated with habitat usage. Longer dive durations and use of the bottom third of the habitat were associated with higher enrichment program index values. Dolphins receiving new enrichment on a monthly/weekly schedule also used the bottom third of the habitat more often than those receiving new enrichment on a yearly/year+ schedule. Dolphins that were managed in a group that was split into smaller subgroups during the day and were reunited into one group at night spent less time in the top third of the habitat than those who remained in a single group with consistent members at all times. Dolphins that were managed as subgroups with rotating members but were never united as one group spent less time in the bottom third of the habitat than those who remained in a single group with consistent members at all times. Taken together, the results suggested that management practices, such as enrichment and training programs, played a greater role in how dolphins interacted with their environment relative to the physical characteristics of the habitat.

Accredited zoological facilities are committed to fully understanding the behavioral, mental, and physical needs of each species to continuously improve the welfare of the animals under their professional care and detect when welfare has diminished. In order to accomplish this goal, internally consistent and externally valid indicators of animal welfare are necessary to advance our understanding of the current welfare status of individual animals. Historically, efforts have focused on monitoring visible or observable signs of poor health or problem behavior, but lack of signs or problems does not necessarily demonstrate that an individual animal is thriving. The current study examined fecal hormone metabolite levels and behavior for two species of bottlenose dolphins (Tursiops truncatus and Tursiops aduncus) from 25 different accredited zoological facilities. At the time of the study, all facilities were accredited by the Alliance of Marine Mammal Parks and Aquariums and/or the Association of Zoos and Aquariums. This was part of the multi-institutional study 'Towards understanding of the welfare of cetaceans in zoos and aquariums" commonly referred to as the Cetacean Welfare Study. Behavioral diversity was calculated using the Shannon Diversity Index on species-appropriate behavioral events. Behavioral diversity was compared to the fecal metabolites of cortisol, aldosterone, and the ratio of cortisol to dehydroepiandrosterone (DHEA) as well as the stereotypic behavior of route tracing. Similar to previous studies on other species, there was a significant inverse relationship between behavioral diversity and both fecal cortisol metabolites and route tracing. Additionally, a significant inverse relationship also exists between behavioral diversity and the ratio of fecal cortisol to DHEA metabolites. Behavioral diversity and fecal aldosterone metabolites were not associated. Additional research is still needed to validate behavioral diversity as an indicator of positive animal welfare for bottlenose dolphins and across species. However, based on current results, facilities could utilize behavioral diversity combined with other measures of welfare to more comprehensively evaluate the welfare of bottlenose dolphins.

Bottlenose dolphins are a behaviorally complex, social species that display a variety of social behaviors. Because of this, it is important for zoological facilities to strive to ensure animals display species-appropriate levels of social behavior. The current study is part of the multi-institutional study entitled "Towards understanding the welfare of cetaceans in zoos and aquariums" commonly referred to as the Cetacean Welfare Study. All participating facilities were accredited by the Alliance of Marine Mammal Parks and Aquariums and/or the Association of Zoos and Aquariums. Behavioral data were collected on 47 bottlenose dolphins representing two subspecies, Tursiops truncatus and Tursiops aduncus, at 25 facilities. The social behaviors of group related activity (group active) as well as interacting with conspecifics (interact with conspecific) were examined for their relationships to both animal management factors and habitat characteristics. The behavioral state of group active and the rate of interact with conspecific were both positively related to the frequency of receiving new forms of environmental enrichment. Both were inversely related to the random scheduling of environmental enrichment. Additional results suggested interact with conspecific was inversely related with daytime spatial experience and that males displayed group active more than females. Overall, the results suggested that animal management techniques such as the type and timing of enrichment may be more important to enhance social behavior than habitat characteristics or the size of the habitat. Information gained from this study can help facilities with bottlenose dolphins manage their enrichment programs in relation to social behaviors.

This study reports comprehensive clinical pathology data for hematology, serum, and plasma biochemistry reference intervals for 174 apparently healthy common bottlenose dolphins (Tursiops truncatus) and reference values for 27 Indo-Pacific bottlenose dolphins (Tursiops aduncus), 13 beluga whales (Delphinapterus leucas), and 6 Pacific white-sided dolphins (Lagenorhynchus obliquidens) in zoos and aquariums accredited by the Alliance for Marine Mammal Parks and Aquariums and the Association of Zoos & Aquariums. Blood samples were collected as part of a larger study titled "Towards understanding the welfare of cetaceans in zoos and aquariums" (colloquially called the Cetacean Welfare Study). Two blood samples were collected following a standardized protocol, and two veterinarian examinations were conducted approximately six months apart between July to November 2018 and January to April 2019. Least square means, standard deviations, and 95% confidence intervals were calculated for hematology, serum, and plasma biochemical variables. Comparisons by age, gender, and month revealed statistically significant differences (p < 0.01) for several variables. Reference intervals and values were generated for samples tested at two laboratories for up to 56 hematologic, serum, and plasma biochemical variables. To apply these data, ZooPhysioTrak, an iOS mobile software application, was developed to provide a new resource for cetacean management. ZooPhysioTrak provides species-specific reference intervals and values based on user inputs of individual demographic and sample information. These data provide a baseline from which to compare hematological, serum, and plasma biochemical values in cetaceans in zoos and aquariums.

The goal of the current study was to create reference intervals and values for several common and one potential novel physiological indicators of animal welfare for four species of cetaceans. The subjects included 189 bottlenose dolphins (Tursiops truncatus), 27 Indo-Pacific bottlenose dolphins (Tursiops aduncus), eight Pacific white-sided dolphins (Lagenorhynchus obliquidens), and 13 beluga whales (Delphinapterus leucas) at Alliance of Marine Mammal Parks and Aquariums and/or Association of Zoos and Aquariums accredited facilities. During two sampling time periods between July and November of 2018 and between January and April of 2019, fecal samples were collected weekly for five weeks from all animals. Samples were processed and analyzed using enzyme immunoassay for fecal cortisol, aldosterone, and dehydroepiandrosterone (DHEA) metabolites. Linear mixed models were used to examine demographic and time factors impacting hormone metabolite concentrations. Age, sex, and time of year were all significant predictors for some of the models (p < 0.01). An iOS mobile application ZooPhysioTrak was created for easy access to species-specific reference intervals and values accounting for significant predictors. For facilities without access to this application, additional reference intervals and values were constructed without accounting for significant predictors. Information gained from this study and the use of the application can provide reference intervals and values to make informed management decisions for cetaceans in zoological facilities.

In recent decades, animal welfare science has evolved to utilize a multidisciplinary approach to assess the welfare of animals in accredited zoos and aquariums. Science-based animal welfare assessments have become an essential component of management programs and widespread application is expected by animal care professionals. Management practices for bottlenose dolphins in accredited facilities incorporate several programs that potentially impact animal welfare including environmental enrichment and animal training. Additionally, habitat characteristics, such as the dimensions of the habitat, have been proposed to affect welfare. While accredited facilities are required to meet high standards of care, habitat characteristics and management practices are not standardized across locations. Knowledge and subsequent application of these practices and habitat characteristics can enhance our understanding of factors associated with positive welfare. As part of a larger study of dolphin welfare titled "Towards understanding the welfare of cetaceans in zoos and aquariums" (colloquially called the Cetacean Welfare Study), survey data were collected from 86 bottlenose dolphins in 40 habitats at 38 facilities in seven countries. The major aims of this paper are to provide general descriptive information regarding dolphin management in accredited zoos and aquariums and to provide supplemental context to the other research published from the Cetacean Welfare Study data set. This paper provides a review of current habitat characteristics and management practices at those 38 accredited zoos and aquariums. These data enabled the identification and quantification of how cetacean management practices differed between participating facilities accredited by the Alliance for Marine Mammal Parks and Aquariums and the Association of Zoos & Aquariums. Variables were selected based on their potential association with welfare including the physical habitat, environmental enrichment, and training programs. The variables were also used for subsequent research in this collection of related papers to investigate important connections between potential indicators of welfare and habitat characteristics, environmental enrichment, and training programs.

Temporal trends in plasma concentrations of per- and polyfluoroalkyl substances (PFAS) in free-ranging bottlenose dolphins (Tursiops truncatus) inhabiting two geographic areas: Indian River Lagoon, Florida over the years 2003-2015 and the waters surrounding Charleston, South Carolina over 2003-2013, were examined. Nine PFAS met the inclusion criteria for analysis based on percent of values below level of detection and sampling years. Proportionate percentiles parametric quantile regression assuming lognormal distributions was used to estimate the average ratio of PFAS concentrations per year for each chemical. Plasma concentrations decreased over time for perfluorodecanoate (PFDA), perfluorohexane sulfonate (PFHxS), perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS), and perfluoroundecanoate (PFUnDA) in both locations. Perfluorononanoate (PFNA) decreased with time in Indian River Lagoon dolphins. Perfluorododecanoate (PFDoDA) concentrations significantly increased over time among female Indian River Lagoon dolphins. Regulation and phaseout of specific PFAS groups may have led to the decreasing levels of those PFAS and increasing levels of other replacement PFAS.

Behavioural differences among social groups can arise from differing ecological conditions, genetic predispositions and/or social learning. In the past, social learning has typically been inferred as responsible for the spread of behaviour by the exclusion of ecological and genetic factors. This 'method of exclusion' was used to infer that 'sponging', a foraging behaviour involving tool use in the bottlenose dolphin (Tursiops aduncus) population in Shark Bay, Western Australia, was socially transmitted. However, previous studies were limited in that they never fully accounted for alternative factors, and that social learning, ecology and genetics are not mutually exclusive in causing behavioural variation. Here, we quantified the importance of social learning on the diffusion of sponging, for the first time explicitly accounting for ecological and genetic factors, using a multi-network version of 'network-based diffusion analysis'. Our results provide compelling support for previous findings that sponging is vertically socially transmitted from mother to (primarily female) offspring. This research illustrates the utility of social network analysis in elucidating the explanatory mechanisms behind the transmission of behaviour in wild animal populations.

Homophilous behaviour plays a central role in the formation of human friendships. Individuals form social ties with others that show similar phenotypic traits, independently of relatedness. Evidence of such homophily can be found in bottlenose dolphins ( Tursiops aduncus) in Shark Bay, Western Australia, where females that use marine sponges as foraging tools often associate with other females that use sponges. 'Sponging' is a socially learned, time-consuming behaviour, transmitted from mother to calf. Previous research illustrated a strong female bias in adopting this technique. The lower propensity for males to engage in sponging may be due to its incompatibility with adult male-specific behaviours, particularly the formation of multi-level alliances. However, the link between sponging and male behaviour has never been formally tested. Here, we show that male spongers associated significantly more often with other male spongers irrespective of their level of relatedness. Male spongers spent significantly more time foraging, and less time resting and travelling, than did male non-spongers. Interestingly, we found no difference in time spent socializing. Our study provides novel insights into the relationship between tool use and activity budgets of male dolphins, and indicates social homophily in the second-order alliance composition of tool-using bottlenose dolphins.

Cetacean health may be potentially affected by anthropogenic sound. We have initiated investigations on the effect of low-frequency underwater sound on immunological gene transcript profiles of captive bottlenose dolphins (Tursiops truncatus) using a probe-based quantitative gene expression assay. Six immunologic genes (IL-2Rα, -4, -10, -12, TNFα and IFNγ) were selected for analysis using two validated housekeeping genes (PGK1 and HPRT1) as reference genes. Twenty-four blood samples from six clinically healthy individuals and six blood samples from individuals after sound exposures were available. The gene transcript profile of sound-exposed dolphins was consistent with a stress-induced TH2 shift profile as compared to controls. This study may lead to better understanding of the effects of anthropogenic sound on immune responses of cetaceans.

The vast majority of bacterial diversity lies within phylum-level lineages called "candidate phyla," which lack isolated representatives and are poorly understood. These bacteria are surprisingly abundant in the oral cavity of marine mammals. We employed a genome-resolved metagenomic approach to recover and characterize genomes and functional potential from microbes in the oral gingival sulcus of two bottlenose dolphins (Tursiops truncatus). We detected organisms from 24 known bacterial phyla and one archaeal phylum. We also recovered genomes from two deep-branching, previously uncharacterized phylum-level lineages (here named "Candidatus Delphibacteria" and "Candidatus Fertabacteria"). The Delphibacteria lineage is found in both managed and wild dolphins; its metabolic profile suggests a capacity for denitrification and a possible role in dolphin health. We uncovered a rich diversity of predicted Cas9 proteins, including the two longest predicted Cas9 proteins to date. Notably, we identified the first type II CRISPR-Cas systems encoded by members of the Candidate Phyla Radiation. Using their spacer sequences, we subsequently identified and assembled a complete Saccharibacteria phage genome. These findings underscore the immense microbial diversity and functional potential that await discovery in previously unexplored environments.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dziobak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chemical analysis of exhaled breath metabolites is an emerging alternative to traditional clinical testing for many physiological conditions. The main advantage of breath analysis is its inherent non-invasive nature and ease of sample collection. Therefore, there exists a great interest in further development of this method for both humans and animals. The physiology of cetaceans is exceptionally well suited for breath analysis due to their explosive breathing behavior and respiratory tract morphology. At the present time, breath analysis in cetaceans has very limited practical applications, in large part due to lack of widely adopted sampling device(s) and methodologies that are well-standardized. Here, we present an optimized design and the operating principles of a portable apparatus for reproducible collection of exhaled breath condensate from small cetaceans, such as bottlenose dolphins (Tursiops truncatus). The device design is optimized to meet two criteria: standardized collection and preservation of information-rich metabolomic content of the biological sample, and animal comfort and ease of breath sample collection. The intent is to furnish a fully-benchmarked technology that can be widely adopted by researchers and conservationists to spur further developments of breath analysis applications for marine mammal health assessments.

Targeted environmental monitoring reveals contamination by known chemicals, but may exclude potentially pervasive but unknown compounds. Marine mammals are sentinels of persistent and bioaccumulative contaminants due to their longevity and high trophic position. Using nontargeted analysis, we constructed a mass spectral library of 327 persistent and bioaccumulative compounds identified in blubber from two ecotypes of common bottlenose dolphins (Tursiops truncatus) sampled in the Southern California Bight. This library of halogenated organic compounds (HOCs) consisted of 180 anthropogenic contaminants, 41 natural products, 4 with mixed sources, 8 with unknown sources, and 94 with partial structural characterization and unknown sources. The abundance of compounds whose structures could not be fully elucidated highlights the prevalence of undiscovered HOCs accumulating in marine food webs. Eighty-six percent of the identified compounds are not currently monitored, including 133 known anthropogenic chemicals. Compounds related to dichlorodiphenyltrichloroethane (DDT) were the most abundant. Natural products were, in some cases, detected at abundances similar to anthropogenic compounds. The profile of naturally occurring HOCs differed between ecotypes, suggesting more abundant offshore sources of these compounds. This nontargeted analytical framework provided a comprehensive list of HOCs that may be characteristic of the region, and its application within monitoring surveys may suggest new chemicals for evaluation.

Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans.

Changing ocean health and the potential impact on marine mammal health are gaining global attention. Direct health assessments of wild marine mammals, however, is inherently difficult. Breath analysis metabolomics is a very attractive assessment tool due to its noninvasive nature, but it is analytically challenging. It has never been attempted in cetaceans for comprehensive metabolite profiling. We have developed a method to reproducibly sample breath from small cetaceans, specifically Atlantic bottlenose dolphins (Tursiops truncatus). We describe the analysis workflow to profile exhaled breath metabolites and provide here a first library of volatile and nonvolatile compounds in cetacean exhaled breath. The described analytical methodology enabled us to document baseline compounds in exhaled breath of healthy animals and to study changes in metabolic content of dolphin breath with regard to a variety of factors. The method of breath analysis may provide a very valuable tool in future wildlife conservation efforts as well as deepen our understanding of marine mammals biology and physiology.

Dolphins, as apex predators, can be considered relevant sentinels of the health of marine ecosystems. The creation of 3D cell models to assess in vitro cell-to-cell and cell-to-matrix interactions in environmental-mimicking conditions, is of considerable interest. However, to date the establishment of cetacean 3D culture systems has not yet been accomplished. Thus, in this study, different 3D systems of bottlenose dolphin (Tursiops truncatus) skin fibroblasts have been analyzed. Particularly, novel scaffolds based on hyaluronic acid and ionic-complementary self-assembling peptides such as RGD-EAbuK and EAbuK-IKVAV have been compared to Matrigel. Histological and fluorescent staining, electron microscopy (TEM) analyses and viability assays have been performed and RT-PCR has been used to detect extracellular matrix (ECM) components produced by cells. Results showed that Matrigel induced cells to form aggregates with lower viability and no ECM production compared to the novel scaffolds. Moreover, scaffolds allowed dispersed cells to produce a collagenous ECM containing collagen1a1, laminin B1 and elastin. The HA-EAbuK-IKVAV scaffold resulted in the most suitable 3D model in terms of cell quantity and viability. The development of this innovative approach is the first step towards the possibility to create 3D in vitro models for this protected species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferretti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events.

Since late 2021, highly pathogenic avian influenza (HPAI) viruses of A/goose/Guangdong/1/1996 (H5N1) lineage have caused widespread mortality in wild birds and poultry in the United States. Concomitant with the spread of HPAI viruses in birds are increasing numbers of mammalian infections, including wild and captive mesocarnivores and carnivores with central nervous system involvement. Here we report HPAI, A(H5N1) of clade 2.3.4.4b, in a common bottlenose dolphin (Tursiops truncatus) from Florida, United States. Pathological findings include neuronal necrosis and inflammation of the brain and meninges, and quantitative real time RT-PCR reveal the brain carried the highest viral load. Virus isolated from the brain contains a S246N neuraminidase substitution which leads to reduced inhibition by neuraminidase inhibitor oseltamivir. The increased prevalence of A(H5N1) viruses in atypical avian hosts and its cross-species transmission into mammalian species highlights the public health importance of continued disease surveillance and biosecurity protocols., (© 2024. The Author(s).)

Passive acoustic monitoring has improved our understanding of vocalizing organisms in remote habitats and during all weather conditions. Many vocally active species are highly mobile, and their populations overlap. However, distinct vocalizations allow the tracking and discrimination of individuals or populations. Using signature whistles, the individually distinct calls of bottlenose dolphins, we calculated a minimum abundance of individuals, characterized and compared signature whistles from five locations, and determined reoccurrences of individuals throughout the Mid-Atlantic Bight and Chesapeake Bay, USA. We identified 1,888 signature whistles in which the duration, number of extrema, start, end, and minimum frequencies of signature whistles varied significantly by site. All characteristics of signature whistles were deemed important for determining from which site the whistle originated and due to the distinct signature whistle characteristics and lack of spatial mixing of the dolphins detected at the Offshore site, we suspect that these dolphins are of a different population than those at the Coastal and Bay sites. Signature whistles were also found to be shorter when sound levels were higher. Using only the passively recorded vocalizations of this marine top predator, we obtained information about its population and how it is affected by ambient sound levels, which will increase as offshore wind energy is developed. In this rapidly developing area, these calls offer critical management insights for this protected species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fandel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dolphins have become famous for their ability to perform a wide variety of athletic and acrobatic behaviors including high-speed swimming, maneuverability, porpoising and tail stands. Tail stands are a behavior where part of the body is held vertically above the water's surface, achieved through thrust produced by horizontal tail fluke oscillations. Strong, efficient propulsors are needed to generate the force required to support the dolphin's body weight, exhibiting chordwise and spanwise flexibility throughout the stroke cycle. To determine how thrust production, fluke flexibility and tail stroke kinematics vary with effort, six adult bottlenose dolphins (Tursiops truncatus) were tested at three different levels based on the position of the center of mass (COM) relative to the water's surface: low (COM below surface), medium (COM at surface) and high (COM above surface) effort. Additionally, fluke flexibility was measured as a flex index (FI=chord length/camber length) at four points in the stroke cycle: center stroke up (CU), extreme top of stroke (ET), center stroke down (CD) and extreme bottom of stroke (EB). Video recordings were analyzed to determine the weight supported above the water (thrust production), peak-to-peak amplitude, stroke frequency and FI. Force production increased with low, medium and high efforts, respectively. Stroke frequency also increased with increased effort. Amplitude remained constant with a mean 33.8% of body length. Significant differences were seen in the FI during the stroke cycle. Changes in FI and stroke frequency allowed for increased force production with effort, and the peak-to-peak amplitude was higher compared with that for horizontal swimming., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The balance between energetic costs and acquisition in free-ranging species is essential for survival, and provides important insights regarding the physiological impact of anthropogenic disturbances on wild animals. For marine mammals such as beluga whales (Delphinapterus leucas), the first step in modeling this bioenergetic balance requires an examination of resting and active metabolic demands. Here, we used open-flow respirometry to measure oxygen consumption during surface rest and submerged swimming by trained beluga whales, and compared these measurements with those of a commonly studied odontocete, the Atlantic bottlenose dolphin (Tursiops truncatus). Both resting metabolic rate (3012±126.0 kJ h-1) and total cost of transport (1.4±0.1 J kg-1 m-1) of beluga whales were consistent with predicted values for moderately sized marine mammals in temperate to cold-water environments, including dolphins measured in the present study. By coupling the rate of oxygen consumption during submerged swimming with locomotor metrics from animal-borne accelerometer tags, we developed predictive relationships for assessing energetic costs from swim speed, stroke rate and partial dynamic acceleration. Combining these energetic data with calculated aerobic dive limits for beluga whales (8.8 min), we found that high-speed responses to disturbance markedly reduce the whale's capacity for prolonged submergence, pushing the cetaceans to costly anaerobic performances that require prolonged recovery periods. Together, these species-specific energetic measurements for beluga whales provide two important metrics, gait-related locomotor costs and aerobic capacity limits, for identifying relative levels of physiological vulnerability to anthropogenic disturbances that have become increasingly pervasive in their Arctic habitats., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Certain deep-diving marine mammals [i.e., northern elephant seal ( Mirounga angustirostris ), Weddell seal ( Leptonychotes weddellii )] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin ( Tursiops truncatus ) and deep-diving beluga whale ( Delphinapterus leucas ), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds. NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

Timely detection and understanding of causes for population decline are essential for effective wildlife management and conservation. Assessing trends in population size has been the standard approach, but we propose that monitoring population health could prove more effective. We collated data from 7 bottlenose dolphin (Tursiops truncatus) populations in the southeastern United States to develop a method for estimating survival probability based on a suite of health measures identified by experts as indices for inflammatory, metabolic, pulmonary, and neuroendocrine systems. We used logistic regression to implement the veterinary expert system for outcome prediction (VESOP) within a Bayesian analysis framework. We fitted parameters with records from 5 of the sites that had a robust network of responders to marine mammal strandings and frequent photographic identification surveys that documented definitive survival outcomes. We also conducted capture-mark-recapture (CMR) analyses of photographic identification data to obtain separate estimates of population survival rates for comparison with VESOP survival estimates. The VESOP analyses showed that multiple measures of health, particularly markers of inflammation, were predictive of 1- and 2-year individual survival. The highest mortality risk 1 year following health assessment related to low alkaline phosphatase (odds ratio [OR] = 10.2 [95% CI: 3.41-26.8]), whereas 2-year mortality was most influenced by elevated globulin (OR = 9.60 [95% CI: 3.88-22.4]); both are markers of inflammation. The VESOP model predicted population-level survival rates that correlated with estimated survival rates from CMR analyses for the same populations (1-year Pearson's r = 0.99, p = 1.52 × 10 -5 ; 2-year r = 0.94, p = 0.001). Although our proposed approach will not detect acute mortality threats that are largely independent of animal health, such as harmful algal blooms, it can be used to detect chronic health conditions that increase mortality risk. Random sampling of the population is important and advancement in remote sampling methods could facilitate more random selection of subjects, obtainment of larger sample sizes, and extension of the approach to other wildlife species., (© 2023 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Finding solutions for a sustainable coexistence between wildlife and humans is considered among the most challenging environmental management issues for scientists, conservationists, managers, and stockholders world-wide. Depredation by the common bottlenose dolphin (Tursiops truncatus) on small scale fisheries has increased in the recent years, leading to a growing conflict in many areas of the Mediterranean Sea and pressing for urgent management solutions. This study aims at developing a management framework for a sustainable coexistence between fishers and dolphins in Sardinia (Mediterranean Sea). Relying on the combination of different approaches (field study, literature review and Multi Criteria Decision Analysis), the scientific evidence necessary for understanding dolphin depredation were updated and improved, the related economic damage was calculated, and different management options were identified and evaluated by several stakeholder groups to support the decision-making process. Averaging for all investigated net types (gillnet and trammel net), a depredation frequency of 53% was found, the highest values ever found in both Sardinia and many other Mediterranean sites. Depredation probability was influenced by different factors, such as net type, fishing operation duration, depth of the fishing site and period. The estimated economic damage due to depredation ranges on average between 6492 and 11,925 euro per year and depends on the type of fishing net. The results from the field study, the literature review and the stakeholder involvement allowed us to define the most plausible and shared management options, identifying a framework for assessing and managing the conflict between fishers and dolphins for the creation of a more sustainable vision for the future., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Nicola Arrostuto, Nicola Fois reports financial support was provided by Autonomous Region of Sardinia., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cetaceans are capable of extraordinary locomotor behaviors in both water and air. Whales and dolphins can execute aerial leaps by swimming rapidly to the water surface to achieve an escape velocity. Previous research on spinner dolphins demonstrated the capability of leaping and completing multiple spins around their longitudinal axis with high angular velocities. This prior research suggested the slender body morphology of spinner dolphins together with the shapes and positions of their appendages allowed for rapid spins in the air. To test whether greater moments of inertia reduced spinning performance, videos and biologging data of cetaceans above and below the water surface were obtained. The principal factors affecting the number of aerial spins a cetacean can execute were moment of inertia and use of control surfaces for subsurface corkscrewing. For spinner dolphin, Pacific striped dolphin, bottlenose dolphin, minke whale and humpback whale, each with swim speeds of 6-7 m s-1, our model predicted that the number of aerial spins executable was 7, 2, 2, 0.76 and 1, respectively, which was consistent with observations. These data implied that the rate of subsurface corkscrewing was limited to 14.0, 6.8, 6.2, 2.2 and 0.75 rad s-1 for spinner dolphins, striped dolphins, bottlenose dolphins, minke whales and humpback whales, respectively. In our study, the moment of inertia of the cetaceans spanned a 21,000-fold range. The greater moments of inertia for the last four species produced large torques on control surfaces that limited subsurface corkscrewing motion and aerial maneuvers compared with spinner dolphins., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The dynamics and drivers of inter-species interactions in the wild are poorly understood, particularly those involving social animal species. Inter-species interactions between cetaceans and sirenians have rarely been documented and investigated. Here, we report 10 cases of interaction initiated by adult bottlenose dolphins (Tursiops truncatus) towards Antillean manatee (Trichechus manatus manatus). Interactions were documented through behavioral observations in the wild (n = 7) and from the examination of orphaned calves (i.e., tooth rake marks on their body; n = 4) that entered a rehabilitation facility, one individual both observed interacting with dolphins and found stranded with bite marks. Bottlenose dolphins were observed interacting with orphan manatee calves and with mother-calf pairs, exhibiting agonistic behavior (n = 2), affiliative or neutral behaviors (n = 1), but the behavioral contexts of these interactions remain unclear in most cases (n = 7). Information on stranded individuals was collected from four calves (of 13 examined calves) recovered in poor condition with bottlenose dolphin tooth rakes and bite wounds on their bodies, one of which died. Injury from bite wounds varied in extent and severity, ranging from superficial scratches leaving rake marks to deep lacerations. Our findings suggest the regular occurrence of agonistic behaviors initiated by adult bottlenose dolphins and directed toward manatee calves. However, the drivers of these interactions remain unknown and need to be further investigated., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Platelet functionality plays a crucial role in marine mammals. Alterations in platelet function can result from stress, pathologies, or exposure to xenobiotics, among others. The early detection of platelet function abnormalities is essential in these species to prevent advanced pathology and mitigate potential risks. Our main objective was to establish a range of physiological values of platelet function in bottlenose dolphins (Tursiops truncatus), beluga whales (Delphinapterus leucas), sea lions (Otaria flavescens) and walruses (Odobenus rosmarus). Intraplatelet Ca 2+ mobilization using adenosine diphosphate (ADP) as a platelet agonist was used as a platelet function biomarker, adapting the methodology previously described by us in dolphins (Felipo-Benavent et al., 2022) to the rest of the species. The assay was also adapted to a seal (Phoca vitulina). Numerical indicators of intraplatelet Ca 2+ mobilization kinetics were established, and statistical analyses were performed to compare the effects of species, sex, age, aquarium and species. Significant differences were observed between species, being the platelets of the sea lions the more reactive to the agonist. This work demonstrates the usefulness of this assay in the diagnosis or monitoring of animals with hemostatic diseases, showing two clinical cases in which intraplatelet calcium mobilization values were altered in marine mammals suffering haemorrhages. This assay may also serve as a means to monitor environmental changes and their potential impact on the health of marine mammal populations., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Echolocating bats and dolphins use biosonar to determine target range, but differences in range discrimination thresholds have been reported for the two species. Whether these differences represent a true difference in their sensory system capability is unknown. Here, the dolphin's range discrimination threshold as a function of absolute range and echo-phase was investigated. Using phantom echoes, the dolphins were trained to echo-inspect two simulated targets and indicate the closer target by pressing a paddle. One target was presented at a time, requiring the dolphin to hold the initial range in memory as they compared it to the second target. Range was simulated by manipulating echo-delay while the received echo levels, relative to the dolphins' clicks, were held constant. Range discrimination thresholds were determined at seven different ranges from 1.75 to 20 m. In contrast to bats, range discrimination thresholds increased from 4 to 75 cm, across the entire ranges tested. To investigate the acoustic features used more directly, discrimination thresholds were determined when the echo was given a random phase shift (±180°). Results for the constant-phase versus the random-phase echo were quantitatively similar, suggesting that dolphins used the envelope of the echo waveform to determine the difference in range.

Importance: Brucella spp. are zoonotic pathogens that can affect both terrestrial and marine mammals. Brucella ceti has been identified in various cetacean species, but only one sequence type (ST27) has been reported in humans. However, it is important to conduct surveillance studies to better understand the impact of marine Brucella species on marine mammals, a typically understudied host group. Here, we describe a systemic infection by two related strains of Brucella pinnipedialis (ST25) in a couple of live-stranded bottlenose dolphins, with more severe lesions in the younger animal. Furthermore, B. pinnipedialis was first detected in milk from a female cetacean that stranded with its offspring. Our study reveals novel insights into the epidemiology and pathological consequences of B. pinnipedialis infections in cetaceans, emphasizing the crucial importance of ongoing surveillance and accurate diagnosis to understand the impact of this pathogen on marine mammal populations., Competing Interests: The authors declare no conflict of interest.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses infect numerous non-human species. Spillover of SARS-CoV-2 into novel animal reservoirs may present a danger to host individuals of these species, particularly worrisome in populations already endangered or threatened by extinction. In addition, emergence in new reservoirs could pose spillback threats to humans, especially in the form of virus variants that further mutate when infecting other animal hosts. Previous work suggests beluga whales ( Delphinapterus leucas ) and bottlenose dolphins ( Tursiops truncatus ) may be at risk owing to their formation of social groups, contact with humans, exposure to contaminated wastewater, and structure of their angiotensin-converting enzyme 2 (ACE2) proteins, which SARS-CoV-2 uses as a cellular receptor. We examined marine-mammal susceptibility to virus infection by challenging 293T cells expressing beluga or dolphin ACE2 with pseudovirions bearing the SARS-CoV-2 spike protein. Beluga and dolphin ACE2 were sufficient to allow cell entry by an early pandemic isolate (Wuhan-Hu-1) and two evolved variants (Delta B.1.617.2 and Omicron BA.1 strains). We conclude that SARS-CoV-2 poses a potential threat to marine mammal reservoirs that should be considered in surveillance efforts.

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

We diagnosed fatal Erysipelothrix rhusiopathiae sepsis in 3 stranded bottlenose dolphins (Tursiops truncatus) during summer 2022, in San Diego, California, USA. The previously undetected disease in this relatively small, regional population of dolphins most likely indicates an environmental or biological change in the coastal ocean or organisms.