Abstract
There is currently a need for health indicators that can be collected from whales by using non-invasive methods to support their conservation efforts. There is also a growing necessity for increased diagnostic abilities and health monitoring in aquarium settings for early disease detection and treatment. Analysis of exhaled breath condensate has been established as a non-invasive method for studying the composition of airway lining fluid with the potential to identify health and inflammation related biomarkers such as cytokines.1 Blow samples from belugas can be collected non-invasively without imposing additional stress,2 however, protocols involving quantification of cytokines and/or chemokines from breath by using immunoassays has been problematic due to their low concentrations, which are often at the lower limits of assay detection.
To address these challenges, quantitative PCR (qPCR) protocols were developed involving gene expression analysis of targeted biomarkers in cells recovered from blow for assessment of respiratory and systemic health status. The feasibility of carrying out sex determination, gene expression and hormone measurements in the same blow sample was also investigated in order to maximize the health information obtained. Targeted biomarkers for gene expression included inflammatory cytokine tumor necrosis-factor-alpha (TNFα); chemokines interleukin-8 (IL8) and C-C-Motif-Chemokine-Ligand-5 (CCL5/RANTES); inflammatory enzyme cyclooxygenase-2 (COX2); and regulatory molecules transforming growth-factor-beta (TGFβ) and glucocorticoid receptor (Nr3c1). Ribosomal protein S9 (RPS9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes to normalize the target gene expression values.
Weekly blow samples were collected into 50 ml conical tubes with DNA/RNA Shield (Zymo Research) throughout the year 2019 in order to determine natural variation. Blow samples were also collected before and after various stressor events (i.e., hydraulic beluga lift, whale transport). DNA, RNA and protein were simultaneously extracted from a small subset of blow samples. Sex determination was carried out using DNA through conventional PCR amplification of zinc-finger genes on X and Y chromosomes (Zfx and Zfy).3 Total RNA was extracted from the entire dataset and gene expression measurements were carried out utilizing two-step SYBR Green qPCR protocols with species-specific primers previously developed in our laboratories.4 The results demonstrated that both DNA (60–375 ng) and RNA (75–5700 ng) can reliably be obtained, and sex determination and gene expression measurements of 4–7 genes can successfully be carried out in the same blow sample depending on yield. The transported whale showed large significant changes in gene expression before vs after transport (>4-fold, p<0.005) for TGFβ, IL8 and Nr3c1 genes, whereas the resident whales displayed variable responses. While resident whale-1 displayed a significant increase in COX2, TGFβ, IL8 and Nr3c1 (p<0.002) following transport along with disturbed behavior, whale-2 did not display any significant changes in gene expression for the markers listed here during the same time period. These target genes are suggested as promising biomarkers of health and the response to stressors in beluga blow. The methodology can potentially be applied to managed-care and wild beluga populations as sensitive tools to aid in research and health assessment.
Acknowledgements
The authors thank the Mystic Aquarium Arctic Coast, husbandry and veterinary teams for their valuable assistance and cooperation in sample collection. The authors acknowledge research intern Alicia Cotoia for helping with sample collection, database organization, RNA extractions and qPCR protocols. In addition, the authors thank Mystic Aquarium research intern Allison Stift and Research for Undergraduate Experiences (REU) student Tonia Osborne for sample collection and processing. Funding for this study was graciously provided by Office of Naval Research (ONR Award No: N00012-18-1-2779), SeaWorld & Busch Gardens Conservation Fund (2018 Session II) and National Science Foundation (NSF # 1658663).
*Presenting author
Literature Cited
1. Liang Y, Yeligar SM, and Brown LAS. 2012. Exhaled breath condensate: a promising source for biomarkers of lung disease. Sci World J 2012(11):217518.
2. Thompson LA, Spoon TR, Goertz CE, Hobbs RC, and Romano TA. 2014. Blow collection as a non-invasive method for measuring cortisol in the Beluga (Delphinapterus leucas). PLoS One 9(12):1–22.
3. Shaw CN, Wilson PJ, White BN. 2003. A reliable molecular method of gender determination for mammals. J Mammal 84(1):123–128.
4. Unal E, Goertz CEC, Hobbs RC, Suydam R, Romano T. 2018. Investigation of molecular biomarkers as potential indicators of health in wild belugas (Delphinapterus leucas). Mar Biol 165:182.