Belugas play a key role in the ecosystem as top predators and sentinels, as well as in the cultural identity of many native peoples of the Arctic. Non-invasive research with trained whales under professional care is a way to develop and validate health-monitoring methods for transition and application to wild whales while helping to inform conservation and management decisions. One potential non-invasive health-monitoring method involves breath or blow, in which hormones, microbes, and molecular components such as expression of health-related immune system genes can be measured. Further, blow sampling has the potential to be utilized with drone technology for sampling of wild whales. The purpose of this project was to further investigate (1) the distance from the blowhole in which a molecular signal can be detected and (2) the relationship between gene expression in blood and blow.

Blow samples were collected from two belugas, one male and one female, aged 19 and 40 years respectively, at Mystic Aquarium, trained to exhale on cue. For objective 1, samples were collected at 2, 4, 8, 12, and 16 inches above the blowhole, then DNA and RNA were simultaneously extracted and quantified using the protocols developed at Mystic Aquarium laboratories. For objective 2, paired blood and blow samples were taken over the span of one year and interleukin-8 (IL8, a target inflammatory gene) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, a reference gene) were quantified by real-time quantitative PCR (qPCR) using a two-step SYBR Green quantification protocol.

DNA and RNA were successfully extracted from each height and determined to be of host origin (i.e., beluga) using PCR amplification with species-specific primers. The results indicated that the DNA yield obtained from both whales was significantly higher at 2 inches versus 8, 12, or 16 inches based on pairwise comparisons (Wilcoxon test, p<0.05). While RNA yield was significantly higher at 2 inches versus 16 inches for whale 1, there were no statistically significant pairwise differences between 2 inches and any other height for whale 2. Interleukin-8 gene expression over the course of the year in both blood and blow was variable, with IL8 showing lower expression in blood (average log₂ fold change of −2.15 from the mean) than in blow (average log₂ fold change of 2.42 from the mean). Gene expression of IL8 in blood and blow was found to be weakly correlated for individual whales (Pearson’s r = 0.31 for whale 1, and r = −0.36 for whale 2).

Future studies will investigate higher distances from the blow hole and examine the relationship between blood and blow using additional genes. Blow shows promise as a non-invasive health-monitoring tool for aiding in the conservation management of wild beluga stocks.

Acknowledgements

The authors thank the Mystic Aquarium animal care and husbandry teams for assisting in the sample collection that allowed this project to happen, and the Mystic Aquarium research team for facilitation. Funding for this study was provided by the National Science Foundation (NSF #1950480 and #1950415).