Project description:Urinary proteins provide valuable insights into renal health, with implications spanning human and domestic animal veterinary medical research. However, the field of marine mammal medicine lacks comprehensive studies on urine protein composition. This research aimed to fill this gap by 1) selecting an optimal search strategy that yields the highest number of protein families in the bottlenose dolphin urine based on post-translational modifications (PTMs), 2) describing the urine proteome of wild bottlenose dolphins (Tursiops truncatus) in the Gulf of the Mexico, USA, considering sex (females vs. males), site (Barataria Bay, LA vs. Sarasota Bay, FL), and comparing with California sea lions (Zalophus californianus). Ten urine samples (Barataria Bay, LA: N= 6; Sarasota Bay, FL: N=4) collected during 2023 catch-and-release heath assessment were proteolytically digested and analyzed using an UltiMate 3000 Nano LC and Fusion Lumos Orbitrap mass spectrometer. A 2-step search strategy, incorporating dehydroalanine formation and semi-trypsin on the list of unassigned spectra, significantly increased the number of identified protein families by an average of 6.2% compared to the 1-step strategy (P < 0.001, t = -8.32). The top 30 proteins in bottlenose dolphin urine were ranked according to an exponentially modified protein abundance index for comparison based on sex, site, and species. There were no significant differences in urine proteins between sexes or sites (padj > 0.05), although there was sperm contribution in two of the male bottlenose dolphin urine samples. Two putative antimicrobial proteins (cathelicidin and lysozyme) were identified and found to be abundant in bottlenose dolphin urine, similar to California sea lions. The study also identified 27 potential markers of acute kidney injury and 12 regulators of kidney stone formation. This study established a reference database of urinary proteins from bottlenose dolphins, aiding future research in monitoring and evaluating renal health in marine mammals.

Project description: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.

Project description:As apex predators, cetaceans are recognized as sentinels for the health of marine ecosystems. The ability to identify the impacts of anthropogenic contamination on the health of a species requires long term monitoring to establish baseline health parameters. This makes health measurements that can be sampled from free ranging animals highly desirable. In the current study, we investigate the utility of skin gene expression profiling to monitor the physiological, health, and contaminant exposure status in bottlenose dolphins. Remote integument biopsies were obtained from bottlenose dolphins at three locations in the northern Gulf of Mexico prior to oil exposure (May 2010) and during summer and winter of the two years (2010-2011) following their exposure to oil from the Deepwater Horizon oil spill. A bottlenose dolphin-specific oligonucleotide microarray was used to characterize the skin transcriptomes of 94 individuals from three genetically distinguishable populations in Barataria Bay, Louisiana, Chandeleur Sound, Louisiana, and Mississippi Sound, Mississippi. Location did not significantly affect the transcriptome patterns of the dolphins included in the study. In contrast, season had a profound effect on gene expression, with nearly one-third of all genes on the array differing significantly in expression between winter and the warmer seasons (p<0.01, fold-change >1.5). Gene ontology enrichment analysis revealed that processes related to cell proliferation, motility and differentiation dominated the seasonal differences in expression, which likely reflects the differences in ambient temperature to which the skin is exposed, and possibly changes in blood flow. More subtle differences were seen between spring and summer transcriptomes (1.5% differentially expressed), with two presumed oil-exposed animals presenting gene expression profiles more similar to the summer (exposed) animals than the other spring animals. Xenobiotic pathway components also showed some seasonal trends, which may reflect crosstalk between xenobiotic pathways and those regulating corticoids, hormones, and cytokines. Seasonal effects have not previously been considered in studies assessing gene expression in cetaceans, but clearly must be taken into account when applying transcriptomic analyses to investigate their exposure or health status.

Project description:Common bottlenose dolphins serve as sentinels for the health of their coastal environments as they are susceptible to health impacts from anthropogenic inputs through both direct exposure and food web magnification. Remote biopsy samples have been widely used to reveal contaminant burdens in free-ranging bottlenose dolphins, but do not address the health consequences of this exposure. To gain insight into whether remote biopsies can also identify health impacts associated with contaminant burdens, we employed RNA sequencing (RNA-seq) to interrogate the transcriptomes of remote skin biopsies from 116 bottlenose dolphins from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts. Gene expression was analyzed using principal component analysis, differential expression testing, and gene co-expression networks, and the results correlated to season, location, and contaminant burden. Season had a significant impact, with over 30% of genes differentially expressed between spring/summer and winter months. Geographic location exhibited lesser effects on the transcriptome, with 15% of genes differentially expressed between the northern Gulf of Mexico and the southeastern U.S. Atlantic locations. Despite a large overlap between the seasonal and geographical gene sets, the pathways altered in the observed gene expression profiles were somewhat distinct. Co-regulated gene modules and differential expression analysis both identified epidermal development and cellular architecture pathways to be expressed at lower levels in animals from the northern Gulf of Mexico. Although contaminant burdens measured were not significantly different between regions, some correlation with contaminant loads in individuals was observed among co-expressed gene modules, but these did not include classical detoxification pathways. Instead, this study identified other, possibly downstream pathways, including those involved in cellular architecture, immune response, and oxidative stress, that may prove to be contaminant responsive markers in bottlenose dolphin skin.

Project description:Considerable efforts have been made to better understand the immune system of bottlenose dolphins in view of the common environmental challenges they encounter, such as exposure to polychlorinated biphenyls, oil spills, or harmful algal bloom biotoxins. However, little is known about the identity and functionality of the Th1, Th2, and Treg T helper cell subsets in bottlenose dolphins. The present study aimed at validating assays and reagents to identify T helper cell subsets and their functions in a subset of dolphins from Sarasota Bay, Florida, USA, which have been long studied and often used as a reference population. A population of CD4+ FOXP3+ lymphocytes was identified representing an average <1% of blood lymphocyte population, which is in the range observed in for Treg cells in other species. The use of porcine reagents to measure TGFß, one of the key Treg cytokines, was further validated using the relatively high-throughput and highly standardized Luminex technology. The proportion of circulating Treg cells was not correlated with the serum concentrations of the Treg effector cytokines TGFß and IL-10, nor could it significantly contribute to predicting the variability of T lymphocyte proliferation, suggesting that not all dolphin circulating Treg cells are functional and active. However, stimulation of dolphin lymphocytes with TGFß and IL-2 increased the expression of the gene for TGFß and IL-10, and stimulation with IL-12 and IFN? induced a robust increase in the expression of the gene for IFN?, suggesting the potential for polarization and differentiation of dolphin T helper cells toward a Treg and Th1 response, respectively. The lack of an increase in the expression of the genes for the Th2 cytokines IL-4 and IL-13 upon stimulation with IL-4 may be due to the requirement for IL-2 for a Th2 polarization as described in mice. However, regression analysis and PCA suggested the potential ability of both the Th1 and Th2 response to be triggered upon acute inflammatory signals. These results may be useful in better understanding the mechanisms by which the dolphin immune system is affected upon exposure to environmental challenges and how it responds to pathogen challenges.

Project description:Inshore common bottlenose dolphins (Tursiops truncatus) are exposed to a broad spectrum of natural and anthropogenic stressors. In response to these stressors, the mammalian adrenal gland releases hormones such as cortisol and aldosterone to maintain physiological and biochemical homeostasis. Consequently, adrenal gland dysfunction results in disruption of hormone secretion and an inappropriate stress response. Our objective herein was to develop diagnostic reference intervals (RIs) for adrenal hormones commonly associated with the stress response (i.e., cortisol, aldosterone) that account for the influence of intrinsic (e.g., age, sex) and extrinsic (e.g., time) factors. Ultimately, these reference intervals will be used to gauge an individual's response to chase-capture stress and could indicate adrenal abnormalities. Linear mixed models (LMMs) were used to evaluate demographic and sampling factors contributing to differences in serum cortisol and aldosterone concentrations among bottlenose dolphins sampled in Sarasota Bay, Florida, USA (2000-2012). Serum cortisol concentrations were significantly associated with elapsed time from initial stimulation to sample collection (p<0.05), and RIs were constructed using nonparametric methods based on elapsed sampling time for dolphins sampled in less than 30 minutes following net deployment (95% RI: 0.91-4.21 µg/dL) and following biological sampling aboard a research vessel (95% RI: 2.32-6.68 µg/dL). To examine the applicability of the pre-sampling cortisol RI across multiple estuarine stocks, data from three additional southeast U.S. sites were compared, revealing that all of the dolphins sampled from the other sites (N = 34) had cortisol concentrations within the 95th percentile RI. Significant associations between serum concentrations of aldosterone and variables reported in previous studies (i.e., age, elapsed sampling time) were not observed in the current project (p<0.05). Also, approximately 16% of Sarasota Bay bottlenose dolphin aldosterone concentrations were below the assay's detection limit (11 pg/mL), thus hindering the ability to derive 95th percentile RIs. Serum aldosterone concentrations from animals sampled at the three additional sites were compared to the detection limit, and the proportion of animals with low aldosterone concentrations was not significantly different than an expected prevalence of 16%. Although this study relied upon long-term, free-ranging bottlenose dolphin health data from a single site, the objective RIs can be used for future evaluation of adrenal function among individuals sampled during capture-release health assessments.

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Blood Transcriptome analysis of wild common bottlenose dolphins (Tursiops truncatus) from the South-East of the United States

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