Pinniped Ontogeny as a Window into the Comparative Physiology and Genomics of Hypoxia Tolerance.

Diving physiology has received considerable scientific attention as it is a central element of the extreme phenotype of marine mammals. Many scientific discoveries have illuminated physiological mechanisms supporting diving, such as massive, internally bound oxygen stores and dramatic cardiovascular regulation. However, the cellular and molecular mechanisms that support the diving phenotype remain mostly unexplored as logistic and legal restrictions limit the extent of scientific manipulation possible. With next-generation sequencing (NGS) tools becoming more widespread and cost-effective, there are new opportunities to explore the diving phenotype. Genomic investigations come with their own challenges, particularly those including cross-species comparisons. Studying the regulatory pathways that underlie diving mammal ontogeny could provide a window into the comparative physiology of hypoxia tolerance. Specifically, in pinnipeds, which shift from terrestrial pups to elite diving adults, there is potential to characterize the transcriptional, epigenetic, and posttranslational differences between contrasting phenotypes while leveraging a common genome. Here we review the current literature detailing the maturation of the diving phenotype in pinnipeds, which has primarily been explored via biomarkers of metabolic capability including antioxidants, muscle fiber typing, and key aerobic and anaerobic metabolic enzymes. We also discuss how NGS tools have been leveraged to study phenotypic shifts within species through ontogeny, and how this approach may be applied to investigate the biochemical and physiological mechanisms that develop as pups become elite diving adults. We conclude with a specific example of the Antarctic Weddell seal by overlapping protein biomarkers with gene regulatory microRNA datasets.

Development-specific transcriptomic profiling suggests new mechanisms for anoxic survival in the ventricle of overwintering turtles.

Oxygen deprivation swiftly damages tissues in most animals, yet some species show remarkable abilities to tolerate little or even no oxygen. Painted turtles exhibit a development-dependent tolerance that allows adults to survive anoxia approximately four times longer than hatchlings: adults survive ∼170 days and hatchlings survive ∼40 days at 3°C. We hypothesized that this difference is related to development-dependent differences in ventricular gene expression. Using a comparative ontogenetic approach, we examined whole transcriptomic changes before, during and 5 days after a 20-day bout of anoxic submergence at 3°C. Ontogeny accounted for more gene expression differences than treatment (anoxia or recovery): 1175 versus 237 genes, respectively. Of the 237 differences, 93 could confer protection against anoxia and reperfusion injury, 68 could be injurious and 20 may be constitutively protective. Most striking during anoxia was the main expression pattern of all 76 annotated ribosomal protein (R-protein) mRNAs, which decreased in anoxia-tolerant adults, but increased in anoxia-sensitive hatchlings, suggesting adult-specific regulation of translational suppression. These genes, along with 60 others that decreased their levels in adults and either increased or remained unchanged in hatchlings, implicate antagonistic pleiotropy as a mechanism to resolve the long-standing question about why hatchling painted turtles overwinter in terrestrial nests, rather than emerge and overwinter in water during their first year. In summary, developmental differences in the transcriptome of the turtle ventricle revealed potentially protective mechanisms that contribute to extraordinary adult-specific anoxia tolerance, and provide a unique perspective on differences between the anoxia-induced molecular responses of anoxia-tolerant and anoxia-sensitive phenotypes within a species.

The effects of pH and Pi on tension and Ca2+ sensitivity of ventricular myofilaments from the anoxia-tolerant painted turtle.

We aimed to determine how increases in intracellular H+ and inorganic phosphate (Pi) to levels observed during anoxic submergence affect contractility in ventricular muscle of the anoxia-tolerant Western painted turtle, Chrysemys picta bellii Skinned multicellular preparations were exposed to six treatments with physiologically relevant levels of pH (7.4, 7.0, 6.6) and Pi (3 and 8 mmol l-1). Each preparation was tested in a range of calcium concentrations (pCa 9.0-4.5) to determine the pCa-tension relationship for each treatment. Acidosis significantly decreased contractility by decreasing Ca2+ sensitivity (pCa50) and tension development (P<0.001). Increasing [Pi] also decreased contractility by decreasing tension development at every pH level (P<0.001) but, alone, did not affect Ca2+ sensitivity (P=0.689). Simultaneous increases in [H+] and [Pi] interacted to attenuate the decreased tension development and Ca2+ sensitivity (P<0.001), possibly reflecting a decreased sensitivity to Pi when it is present as the dihydrogen phosphate form, which increases as pH decreases. Compared with that of mammals, the ventricle of turtles exhibits higher Ca2+ sensitivity, which is consistent with previous studies of ectothermic vertebrates.