A blubber gene expression index for evaluating stress in marine mammals

Abstract

Evaluating the impacts of anthropogenic disturbance on free-ranging marine mammal populations, many of which are in decline, requires robust diagnostic markers of physiological stress and health. However, circulating levels of canonical ‘stress hormones’ such as glucocorticoids, which are commonly used to evaluate animal health, do not capture the complexity of species-specific responses and cannot be easily measured in large, fully aquatic marine mammals. Alternatively, expression of stress-responsive genes in hormone target tissues such as blubber, the specialized subcutaneous adipose tissue that can be manually or remotely sampled from many marine mammals, may be a more informative and sensitive indicator of recent (within 24 h) exposure to stressors. We previously identified genes that were upregulated in the inner blubber of juvenile northern elephant seals during experimental stimulation of the hypothalamic–pituitary–adrenal axis. In this study, we measured baseline expression levels of a subset of these genes in inner blubber of unmanipulated juvenile elephant seals of varying physiological states and correlated them with other stress markers (body condition index, corticosteroid and thyroid hormone levels). Expression of 10 genes, including those associated with lipid metabolism (ACSL1, HMGCS2, CDO1), redox homeostasis (GPX3), adipokine signaling (ADIPOQ), lipid droplet formation (PLIN1, CIDEA) and adipogenesis (DKK1, AZGP1, TGFBI), was described by three principal components and was associated with cortisol and thyroid hormone levels. Significantly, baseline gene expression levels were predictive of circulating hormone levels, suggesting that these markers may be potential indicators of exposure to stressors in marine mammal species that are inaccessible for blood sampling. A similar approach may be used to identify species-specific stress markers in other tissues that can be sampled by remote biopsy dart from free-ranging marine mammals, such as outer blubber and skin.

Ontogeny of Carbon Monoxide-Related Gene Expression in a Deep-Diving Marine Mammal

“…RNA integrity numbers (RIN) for muscle and blood samples were 8.93 ± 0.37 and 5.11 ± 1.26, respectively. RIN values for RNA isolated from muscle samples that were preserved in RNA later TM and those that were flash frozen in liquid nitrogen were comparable as shown previously ( Pujade Busqueta et al, 2020 ).…”

“…RNA later TM was removed from samples prior to freezing at −80°C for long term storage. We previously showed that the integrity of RNA isolated from samples frozen in the field and those preserved in RNA later TM were comparable and can be used interchangeably for targeted gene expression analyses ( Pujade Busqueta et al, 2020 ).…”

“…Muscle biopsies collected from juveniles were preserved in RNAlater TM Stabilizing Solution (∼300 mg tissue per 1.5 mL; Invitrogen, United States) for 24 h at 4 • C. RNAlater TM was removed from samples prior to freezing at −80 • C for long term storage. We previously showed that the integrity of RNA isolated from samples frozen in the field and those preserved in RNAlater TM were comparable and can be used interchangeably for targeted gene expression analyses (Pujade Busqueta et al, 2020).…”

Species and population specific gene expression in blood transcriptomes of marine turtles

“…Relationships between environmental stressors such as pollutant and pathogen exposure and transcriptomic responses have been documented in a wide variety of taxa, including great tits [ 8 ], killifish ( Fundulus heteroclitus [ 73 ]), and wild salmon smolts ( Oncorhynchus nerka [ 74 ]). Additionally, correlations between stress hormones and gene expression have been documented in elephant seals ( Mirounga angustirostris [ 75 ]). Transcriptomic data can also be paired with sex or life stage information to explore the potential for developing gene expression-based biomarkers to determine important demographic information such as sex, which would be a useful addition to the current methods available for sexing immature marine turtles [ 76 , 77 ].…”

Comprehensive molecular and morphological resolution of blubber stratification in a deep-diving, fasting-adapted seal

“…Animals were sedated and sampled as described previously ( Debier et al, 2020 ; Pujade Busqueta et al, 2020 ). Briefly, seals were chemically immobilized using an intramuscular injection of ∼1 mg/kg tiletamine-zolazepam HCl (Telazol, Fort Dodge Animal Health, United States), and sedation was maintained with intravenous doses of ketamine (0.25–1 mg/kg) (Fort Dodge Animal Health, IA, United States).…”