Abstract
Beluga whales (Delphinapterus leucas) live in the Arctic and Subarctic along Alaska, Canada, Russia, Norway, Denmark, and Greenland coasts. Their habitat is ground zero for climate change with exposure to a variety of environmental and anthropogenic stressors. Research on wild beluga populations is necessary for conservation and management. Beluga whales at Mystic Aquarium (Mystic, Connecticut, USA) are critical to the advancements of conservation research. For this study, respiratory exhale (blow) samples were utilized as a non-invasive sampling technique to offer data on sex, immune, and population status. The main objectives of this project were to 1) amplify both sex and mitochondrial DNA (mtDNA) genetic markers in one exhale blow sample; 2) simulate a blow sample collection from a wild beluga using trained aquarium whales to process for these markers; and 3) promote advancements in this technology with a view towards research applications on wild belugas.
Single exhale blow samples were collected from three trained adult beluga whales (1 male, 2 females) at Mystic Aquarium under behavioral control. Blow samples were collected when whales were stationed (clear [sample to clear the blowhole] and one-exhale samples) and, during a free swim simulation, then immediately preserved with DNA/RNA Shield™ (Zymo Research). DNA from blow samples was extracted with a Quick-DNA™ MiniPrep Kit (Zymo Research). Previously published sex primers specific to mammals1,2 and mitochondrial DNA (mtDNA) primers specific to beluga whales3 were used in a polymerase chain reaction (PCR) to amplify for the zinc finger (ZF) sex-linked genes (931–1006 base pairs) and mtDNA (400–700 base pairs), and PCR products were visualized on a 1.5% agarose gel. Amplified mtDNA samples were sent out for sequencing for confirmation, and the resultant sequences were aligned using Bioedit v7.7.1 sequence alignment editor to identify the presence of base-pair differences among individuals.
Total DNA yield across all sampling methods ranged between 172.5 and 1344.5 ng for clear samples, 698 and 946.5 ng for one-exhale samples, and 87 and 196.5 ng for free-swim samples and did not change significantly among these different collection types. The yield was sufficient for sex identification and mtDNA amplification in the same blow sample with a single exhale for all the replicate samples collected. The mtDNA sequences were confirmed as beluga whale for all samples, and base-pair differences were identified in an individual beluga known to originate from a different stock. DNA/RNA Shield™ was confirmed as a valuable preservative to use and apply to wild beluga sampling in field conditions where freezing is not feasible, and blow samples (with a single breath) show promise as a viable non-invasive sampling technique to obtain molecular information on wild belugas. The ability to simultaneously determine the sex and mtDNA sequences in wild belugas has the potential to provide us with valuable information on stock identification, male-to-female ratios in stocks, and conservation needs.
Acknowledgments
The authors would like to thank the beluga whale and research teams at Mystic Aquarium for their support in sample collection for this research. The research was funded by a National Science Foundation (NSF) Research Experiences for Undergraduates (REU) site grant to Mystic Aquarium (Award #1950480) and the University of Connecticut (Award # 1950415).
*Presenting author
+Student presenter
Literature Cited
1. Shaw CN, Wilson PJ, White BN. A reliable molecular method of gender determination for mammals. J Mammal. 2003;84:123–128.
2. Richard JT, Schultz K, Goertz C, Hobbs R, Romano TA, Sartini BL. Assessing the quantity and downstream performance of DNA isolated from beluga (Delphinapterus leucas) blow samples. Aquatic Mammals. 2017;4:398.
3. O'Corry‐Crowe GM, Suydam RS, Rosenberg A, Frost KJ, Dizon AE. Phylogeography, population structure and dispersal patterns of the beluga whale Delphinapterus leucas in the western Nearctic revealed by mitochondrial DNA. Mol Ecol. 1997;6:955–970.