Abstract

The use of blow sampling in cetaceans has gained attention as an alternative to blood for monitoring health in cetaceans via measurement of steroid hormones, genetics and gene expression, and microbiome. Because blow contains particles of mucus and lung fluid, there are other proteins in blow that can offer further biological information about individual animals. In 2018, our laboratory presented changes in serum catecholamines and complement (C5a) in a single beluga following specific active or stationary dive behaviors.¹ We also presented preliminary measures of C5a changes in blow, which displayed similar though much smaller changes as compared with blood—suggesting that complement proteins, as well as other markers of inflammation or immune status, are measurable in blow. The purposes of this work were to provide the first description of the entire protein profile of beluga blow and serum samples and to identify potential biomarkers for future studies. Five paired serum and blow samples from two belugas (one male, 16 years old; one female, 36 years old), as well as pre- and post-blow samples from four dive trails in a single female beluga (two active and two stationary), were analyzed by shotgun proteomics. The protein profiles of beluga blood and blow were compared (Pearson correlation α=0.05). Changes in the protein profile following dive behaviors were visually assessed in order to 1) confirm previous C5a study results and 2) identify protein targets that may be important biomarkers in dive physiology. On average, 718 proteins were identified in blow samples while only 145 were identified in blood. One hundred and sixteen proteins were shared between serum and blow; however, few positive correlations were identified, and most relationships were not significant. The 100 most abundant proteins in blow were determined to contribute to complement and coagulation cascades, estrogen metabolism, platelet function, prolactin signaling, calcium regulation, TNFα and NFкB signaling, glycolysis and gluconeogenesis, and exercise-induced circadian regulation. Preliminary analysis from a single paired active and stationary dive suggests protein abundance decreased pre to post for the active dive, and remained low 1 hour later, while for stationary dives protein abundance recovered 1 hour post behavior. Proteins displaying an increase in abundance immediately following dive behaviors appear to play roles in oxygen and carbon dioxide exchange and hemoglobin stabilization pathways. Vanin-1, which may play a role in tolerance to oxidative stress, was higher in serum following dives as compared with monthly baseline values, and was also detectable in blow. While C5a was not detected in blow, the precursor protein C5 was, and patterns of change following dive behaviors support previous results. Further analysis of samples is ongoing to determine potential biomarkers. This work represents the first report on the protein profile of beluga blow and serum.

Acknowledgements

Funding for collection of samples following dive behaviors was provided by the Office of Naval Research Award #N000141512203 and N00014-18-1-2779. The authors thank the beluga husbandry staff at Mystic Aquarium for the training of beluga dive behaviors, and participation in this study, particularly Carey Richard, Lindsey Nelson, and Kathryn Justice. Thank you to the veterinary team at Mystic Aquarium for carrying out blood sampling. This work was conducted under the Mystic Aquarium IACUC protocol #15004.

*Presenting author

Literature Cited

1.  Thompson LA, Romano TA. 2018. Activity level during diving plays a role in the complement response to nitrogen bubbles in a beluga (Delphinapterus leucas). IAAAM 49th Annual Conference Proceedings, Long Beach, CA.