Abstract

The relationship between the presence of gas bubbles and inflammatory processes, via the complement system, in the development of damage associated with decompression sickness in humans and terrestrial mammals has been demonstrated; and lack of damage from bubbles has been noted where immune responses have failed to be elicited. Little is known about the occurrence of 'silent bubbles' or the role of complement in marine mammal resistance to dive-related injury, but new studies suggest less sensitive immune responses serve a protective role under normal circumstances. For this study, an enzyme immunoassay created with harbor seal proteins was used to measure C5a, a product of complement activation, in serum samples from belugas and harbor seals with and without in vitro exposures to nitrogen bubbles. Samples were obtained from belugas resident at Mystic Aquarium (2009–2016; n=7) and compared to wild animals from Bristol Bay, AK which were sampled during health assessment efforts (2014, 2016; n=9); as well as from stranded harbor seals sampled at the time of admit to rehabilitation at Mystic Aquarium and again at release (2016; n=9). Four flow rates were used for nitrogen bubble exposure: 0.02, 0.06, 0.5 and 1 ml/min. All exposures were done for 30 min. Control measures of C5a were significantly higher in admit harbor seal samples as compared with release (Paired T-test; p=0.013); as well as in aquarium belugas compared to Bristol Bay whales (Mann Whitney U-test; p=0.016). Aquarium belugas showed no significant changes in C5a following N2 bubble exposures, while Bristol Bay animals showed a small but significant increase in C5a only following exposure to nitrogen at a flow rate of 1 ml/min (GLM; p=0.019). Bristol Bay animals also showed a tendency of larger changes in C5a following N2 exposures than aquarium belugas; and a significant positive correlation was detected between control C5a and the bubble induced change in C5a for exposures to 1 ml/min (Pearson; p=0.001). Harbor seals displayed no significant changes in C5a for admit samples, but a significant increase in release samples following exposure to 0.02 ml/min (GLM: p=0.045). Large changes, and variation, were also noted in release samples for flow rates of 0.06 and 0.5 ml/min. For both of these exposures a significant negative correlation was detected between the control C5a and the bubble-induced change in C5a (Pearson; 0.06 ml/min, p=0.04; 0.5 ml/min, p=0.034). This study is the first to measure C5a as a marker of complement activation in response to nitrogen bubbles in marine mammals. The results suggest that circulating C5a may vary based on physiology (e.g., "stressed vs. non-stressed"), and that the complement response to nitrogen bubbles in both belugas and harbor seals is less sensitive than in humans, though there may be some minor differences in response between these species based on dive capability. This data supports the hypothesis of a less reactive immune response serving a protective function in diving mammals, and suggests that silent bubbles may occur in these animals. Further work is needed to understand the mechanism behind this adaptation.

Acknowledgements

The authors would like to thank the Research and Veterinary Services department at the Mystic Aquarium, as well as the beluga husbandry staff and staff of the animal rescue clinic for helping to provide samples. Thank you to the Bristol Bay Field Team for efforts in sampling free ranging belugas, as well as to the Bristol Bay Native Association, Bristol Bay Marine Mammal Council, the Alaska Beluga Whale Committee and the Alaska Department of Fish and Game for permission to conduct such field work. Field efforts were supported by Georgia Aquarium. This work was funded by the Office of Naval Research (ONR) award #N000141512203. Sampling of belugas in Bristol Bay was conducted under NMFS Scientific Research Permit #14245.

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