Environment, endocrinology, and biochemistry influence expression of stress proteins in bottlenose dolphins.

Natural and anthropogenic stressors have been reported to impact the health of marine mammals. Therefore, investigation of quantifiable biomarkers in response to stressors is required. We hypothesized that stress protein expression would be associated with biological and health variables in wild and managed-care bottlenose dolphins (Tursiops truncatus). To test this hypothesis, our study objectives were to (1) determine if stress proteins in skin, white blood cells (WBCs), and plasma could be measured with an antibody-based microarray, (2) measure stress-protein expression relative to biological data (location, sex, age, environment), and (3) determine if stress-protein expression was associated with endocrine, hematological, biochemical and serological variables and gene expression in bottlenose dolphins. Samples were collected from two wild groups (n = 28) and two managed-care groups (n = 17). Proteins involved in the HPA axis, apoptosis, proteotoxicity, and inflammation were identified as stress proteins. The expression of 3 out of 33 proteins was significantly (P < 0.05) greater in skin than plasma and WBCs. Male dolphins had significantly greater expression levels for 10 proteins in skin compared to females. The greatest number of stress-associated proteins varied by the dolphins' environment; nine were greater in managed-care dolphins and 15 were greater in wild dolphins, which may be related to wild dolphin disease status. Protein expression in skin and WBCs showed many positive relationships with measures of plasma endocrinology and biochemistry. This study provides further understanding of the underlying mechanisms of the stress response in bottlenose dolphins and application of a combination of novel methods to measure stress in wildlife.

Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.