Abstract

Changes in the expression of inflammatory and regulatory genes in response to external stressors reflect an early stage in immune system imbalances and can potentially be utilized to help prevent negative health outcomes. Identification of any significant changes in expression of these genes relies on the availability of the baseline data incorporating seasonal, diurnal and/or sex-specific changes. Utilizing non-invasive sample matrices for health monitoring in whales is desired in aquarium settings to complement and maximize the health information that can be gained from the individuals. For cetaceans, exhaled breath condensate (blow), which is composed of the airway lining fluid, is increasingly being used to monitor hormones, to identify molecular components, and for disease surveillance.^1–5

The main objectives of this study were: 1) to investigate the natural variability in gene expression in blood and blow samples collected from two aquarium belugas (Delphinapterus leucas) within one year 2) to investigate potential correlations between blood versus blow gene expression by utilizing paired monthly samples. Molecular assays were developed for the following target genes using real-time quantitative PCR (qPCR): Inflammatory markers tumor-necrosis-factor-alpha (TNFα), interleukin-1β (IL1β), interleukin-8 (IL8), C-C-Motif-Chemokine-Ligand-5 (CCL5/RANTES), and cyclooxygenase-2 (COX2); immune receptor protein toll-like-receptor-4 (TLR4); and regulatory molecule transforming growth-factor-beta (TGFβ). For objective 1, monthly blood and biweekly blow samples were collected between July 2018–July 2019 from two belugas (male and female). For objective 2, monthly paired blow and blood samples obtained from the same whales were utilized. Blow samples were collected into 50 ml conical tubes with DNA/RNA Shield™ (Zymo Research). Blood samples were collected in PAXgene® blood RNA tubes (BD Biosciences). Following total RNA extraction, gene expression measurements were carried out utilizing two-step SYBR Green qPCR protocols with species-specific primers previously developed in our laboratories.⁶

Results indicate significant (ANOVA p<0.01) seasonality in gene expression in both blood (IL8, TGFβ, TNFα, COX2, IL1β, CCL5, TLR4) and blow (IL8, TGFβ, TNFα) samples. While blow samples showed lowest gene expression of targets IL8 and TNFα during winter, blood samples displayed lowest gene expression during spring for IL8, TGFβ, TNFα and COX2. Mann-Whitney tests performed on female versus male blow samples resulted in significantly more expression in the male for IL8 (p<0.01), TGFβ (p<0.05) and COX2 (p<0.05), but not for TNFα. However, there were no significant differences between the sexes for blood samples. IL8 and TNFα showed significantly (p<0.0001) higher expression in blow samples when compared to blood, whereas TGFβ expression was significantly (p<0.0001) lower in blow. No significant correlations were observed between blood and blow for this sampling year for the three genes tested in the combined dataset (Pearson’s r p>0.05), so far excluding the possibility of using blow as an indicator of blood. These differences could potentially be reflecting differences in physiology, including possible effects of the breeding season which takes place anywhere between February and May. Overall, this study contributes towards an understanding of temporal and sex/age specific influences on gene expression while pointing out to the future utilization of blow gene expression for assessment of health status in belugas.

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 interns Alicia Cotoia for helping with blow sample collection, processing and database organization, and Allison Stift for blood RNA extractions. In addition, the authors thank Mystic Aquarium Research for Undergraduate Experiences (REU) student Bailey McKenna for sample processing. Funding for this study was 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).

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