Gene Expression Profiling as a Potential Tool for Health Assessment in Belugas (Delphinapterus leucas)
Gene-based health screening have the potential to reveal changes in physiological status utilizing minimal amounts of tissue sample, and can be used as a source of molecular biomarkers to assess various immune challenges. Moreover, there is currently a need for whale health indicators that can be collected by using non-invasive techniques in order to support their conservation efforts. This study screened gene expression profiles of ten stress and immune-related biomarkers using real-time polymerase chain reaction in both wild and aquarium belugas, reporting their reference ranges for blood and skin. Respiratory exhale (blow) samples collected from aquarium belugas during an acclimation period to a stressor event were also utilized with the potential to identify immune/inflammation related biomarkers in airway lining fluid. Blood (n=109) and skin (n=97) samples were collected from two different populations of wild belugas in Alaska: 1) following live capture-release health assessments during 2008, 2012–2014, 2016 in Bristol Bay, 2) following live-capture-release health assessments and subsistence hunts during 2012–2014, 2016, 2017 in Eastern Chukchi Sea. Blood (n=5) and blow (n=2) samples were also collected from resident aquarium belugas behaviorally. Live-captured whales showed significant (p<0.05) down-regulation of interferon-gamma (IFNγ) pre- versus post-examination blood along with increasing serum cortisol levels, indicative of the physiological response to capture stress. Wild belugas also displayed higher levels of inflammatory IFNγ and stress response marker glucocorticoid receptor (Nr3c1) when compared to aquarium whales. When compared to blood, skin samples displayed higher differentiation among the two wild populations (Bristol Bay and Eastern Chukhi Sea) based on t-tests, principle component analysis, and general linear model analysis including significant (multiple test corrected p<0.05) seasonal and/or spatial effects. Interleukins IL10 and IL12, TGFβ, Nr3c1, toll-like receptor-4 (TLR4), and aryl hydrocarbon receptor (AHR) were the most informative biomarkers in skin reflecting gene expression changes between populations. Out of the markers previously validated in blood, six markers were also successfully amplified in blow samples collected from aquarium whales including transforming growth factor beta (TGFβ), Interleukin 1 beta (IL1β), cyclooxygenase-2 (COX2), Nr3c1, tumor necrosis factor alpha (TNFα) and AHR. In blow, significant (p<0.0001) variation in TGFβ expression was observed between the baseline values of the two whales, however, TGFβ expression remained stable across the sampling time period for each whale. On the other hand, COX2 displayed significant (p<0.0001) variation throughout the sampling time period, however, no significant variation was observed between the two whales for the period tested. Since gene expression studies can capture the changes and imbalances at the cellular level before clinical symptoms develop, proposed methods can potentially be utilized as additional diagnostic and health monitoring tools for aquarium facilities, and can also be applied to health assessment studies of wild/free-ranging whales.
This study was funded by Office of Naval Research (ONR Award no: N00014-14-1-0411) and the SeaWorld & Busch Gardens Conservation Fund (Award No: 1236). The authors thank the Department of Wildlife Management, North Slope Borough, Barrow, AK, the Point Lay field team and the community of Point Lay, AK. The authors also thank the Bristol Bay field team along with the Bristol Bay Marine Mammal Council and Bristol Bay Native Association. The Alaska Beluga Whale Committee encouraged health studies on belugas. In addition, the authors acknowledge the Mystic Aquarium veterinary team, animal care team, the Arctic Coast husbandry team and research staff for their collaboration and assistance in sampling aquarium whales.