Transcriptome analysis of northern elephant seal (Mirounga angustirostris) muscle tissue provides a novel molecular resource and physiological insights

BackgroundThe northern elephant seal, Mirounga angustirostris, is a valuable animal model of fasting adaptation and hypoxic stress tolerance. However, no reference sequence is currently available for this and many other marine mammal study systems, hindering molecular understanding of marine adaptations and unique physiology.ResultsWe sequenced a transcriptome of M. angustirostris derived from muscle sampled during an acute stress challenge experiment to identify species-specific markers of stress axis activation and recovery. De novo assembly generated 164,966 contigs and a total of 522,699 transcripts, of which 68.70% were annotated using mouse, human, and domestic dog reference protein sequences. To reduce transcript redundancy, we removed highly similar isoforms in large gene families and produced a filtered assembly containing 336,657 transcripts. We found that a large number of annotated genes are associated with metabolic signaling, immune and stress responses, and muscle function. Preliminary differential expression analysis suggests a limited transcriptional response to acute stress involving alterations in metabolic and immune pathways and muscle tissue maintenance, potentially driven by early response transcription factors such as Cebpd.ConclusionsWe present the first reference sequence for Mirounga angustirostris produced by RNA sequencing of muscle tissue and cloud-based de novo transcriptome assembly. We annotated 395,102 transcripts, some of which may be novel isoforms, and have identified thousands of genes involved in key physiological processes. This resource provides elephant seal-specific gene sequences, complementing existing metabolite and protein expression studies and enabling future work on molecular pathways regulating adaptations such as fasting, hypoxia, and environmental stress responses in marine mammals

Changes in serum adipokines during natural extended fasts in female northern elephant seals

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Rzucidlo, C. L., Sperou, E. S., Holser, R. R., Khudyakov, J., Costa, D. P., & Crocker, D. E. Changes in serum adipokines during natural extended fasts in female northern elephant seals. General and Comparative Endocrinology, 308, (2021): 113760, https://doi.org/10.1016/j.ygcen.2021.113760.Adipose tissue is essential to endotherms for thermoregulation and energy storage as well as functioning as an endocrine organ. Adipose derived hormones, or adipokines, regulate metabolism, energy expenditure, reproduction, and immune function in model systems but are less well studied in wildlife. Female northern elephant seals (NES) achieve high adiposity during foraging and then undergo natural fasts up to five weeks long during haul-outs associated with reproduction and molting, resulting in large changes in adipose reserves. We measured circulating levels of four adipokines: leptin, resistin, adiponectin, and kisspeptin-54, in 196 serum samples from female NES at the beginning and end of their breeding and molting fasts. We examined the relationships between these adipokines and life-history stage, adiposity, mass, cortisol, and an immune cytokine involved in the innate immune response interleukin 6 (IL-6). All four adipokines varied with life-history stage. Leptin concentrations were highest at the beginning of the breeding haul-out. Resistin concentrations were higher throughout the breeding haul-out compared to the molt haul-out. Adiponectin concentrations were highest at the beginning of both haul-outs. Kisspeptin-54 concentrations were highest at the end of the breeding haul-out. Leptin, resistin, and adiponectin were associated with measures of body condition, either adiposity, mass, or both. Resistin, adiponectin, and kisspeptin-54 were associated with circulating cortisol concentrations. Resistin was strongly associated with circulating IL-6, a multifunctional cytokine. Adiponectin was associated with glucose concentrations, suggesting a potential role in tissue-specific insulin sensitivity during life-history stages categorized by high adiposity. Increased cortisol concentrations late in lactation were associated with increased kisspeptin-54, suggesting a link to ovulation initiation in NES. This study suggests dramatic changes in circulating adipokines with life-history and body condition that may exert important regulatory roles in NES. The positive relationship between adiponectin and adiposity as well as the lack of a relationship between leptin and kisspeptin-54 differed from model systems. These differences from biomedical model systems suggest the potential for modifications of expression and function of adipose-derived hormones in species that undergo natural changes in adiposity as part of their life-history.This project was supported by a grant from the Office of Naval Research (#N00014-18-1-2822) to DPC and DEC and the Marine Life Joint Industry Program of the IAGOP. We thank the Año Nuevo State Reserve rangers for logistical support

Ex vivo and in vitro methods as a platform for studying anthropogenic effects on marine mammals: four challenges and how to meet them

Marine mammals are integral to global biodiversity and marine health through their roles in coastal, benthic, and pelagic ecosystems. Marine mammals face escalating threats from climate change, pollution, and human activities, which perturb their oceanic environment. The diverse biology and extreme adaptations evolved by marine mammals make them important study subjects for understanding anthropogenic pressures on marine ecosystems. However, ethical and logistical constraints restrict the tractability of experimental research with live marine mammals. Additionally, studies on the effects of changing ocean environments are further complicated by intricate gene-environment interactions across populations and species. These obstacles can be overcome with a comprehensive strategy that involves a systems-level approach integrating genotype to phenotype using rigorously defined experimental conditions in vitro and ex vivo. A thorough analysis of the interactions between the genetics of marine mammals and their exposure to anthropogenic pressures will enable robust predictions about how global environmental changes will affect their health and populations. In this perspective, we discuss four challenges of implementing such non-invasive approaches across scientific fields and international borders: 1) practical and ethical limitations of in vivo experimentation with marine mammals, 2) accessibility to relevant tissue samples and cell cultures; 3) open access to harmonized methods and datasets and 4) ethical and equitable research practices. Successful implementation of the proposed approach has the potential impact to inspire new solutions and strategies for marine conservation

Transcriptome analysis of northern elephant seal (Mirounga angustirostris) muscle tissue provides a novel molecular resource and physiological insights.

BackgroundThe northern elephant seal, Mirounga angustirostris, is a valuable animal model of fasting adaptation and hypoxic stress tolerance. However, no reference sequence is currently available for this and many other marine mammal study systems, hindering molecular understanding of marine adaptations and unique physiology.ResultsWe sequenced a transcriptome of M. angustirostris derived from muscle sampled during an acute stress challenge experiment to identify species-specific markers of stress axis activation and recovery. De novo assembly generated 164,966 contigs and a total of 522,699 transcripts, of which 68.70% were annotated using mouse, human, and domestic dog reference protein sequences. To reduce transcript redundancy, we removed highly similar isoforms in large gene families and produced a filtered assembly containing 336,657 transcripts. We found that a large number of annotated genes are associated with metabolic signaling, immune and stress responses, and muscle function. Preliminary differential expression analysis suggests a limited transcriptional response to acute stress involving alterations in metabolic and immune pathways and muscle tissue maintenance, potentially driven by early response transcription factors such as Cebpd.ConclusionsWe present the first reference sequence for Mirounga angustirostris produced by RNA sequencing of muscle tissue and cloud-based de novo transcriptome assembly. We annotated 395,102 transcripts, some of which may be novel isoforms, and have identified thousands of genes involved in key physiological processes. This resource provides elephant seal-specific gene sequences, complementing existing metabolite and protein expression studies and enabling future work on molecular pathways regulating adaptations such as fasting, hypoxia, and environmental stress responses in marine mammals

Expression of obesity-related adipokine genes during fasting in a naturally obese marine mammal

Northern elephant seals ( Mirounga angustirostris) are exceptional among fasting-adapted animals in coupling prolonged fasting with energetically costly activities, relying on oxidation of fat stores accrued during foraging to power metabolic demands of reproduction and molting. We hypothesized that high rates of energy expenditure, insulin resistance, and immune responses to colonial breeding in fasting seals are mediated by adipokines, or signaling molecules secreted by adipose tissue that are associated with obesity and inflammation in humans. We measured mRNA expression of 10 adipokine genes in blubber tissue of adult female elephant seals sampled early and late during their lactation and molting fasts and correlated gene expression with adiposity and circulating levels of corticosteroid and immune markers. Expression of adiponectin ( ADIPOQ) and its receptor ADIPOR2, leptin receptor ( LEPR), resistin ( RETN), retinol binding protein 4 ( RBP4), and visfatin/nicotinamide phosphoribosyltransferase ( NAMPT) was increased, whereas that of fat mass and obesity-associated protein ( FTO) was decreased in late-fasted compared with early-fasted groups. Abundance of adipokine transcripts that increased in late fasting was negatively associated with body mass and positively associated with cortisol, suggesting that they may mediate local metabolic effects of cortisol in blubber during fasting. Expression of several adipokines was correlated with the immune markers IL-6, haptoglobin, IgM, and IgE, suggesting a potential role in modulating immune responses to colonial breeding and molting. Since many of these adipokines have not been measured in other wild animals, this study provides preliminary insights into their local regulation in fat tissue and targeted assays for future studies.</jats:p

Liver proteome response to torpor in a basoendothermic mammal, <i>Tenrec ecaudatus</i>, provides insights into the evolution of homeothermy

Many mammals use adaptive heterothermy (e.g., torpor, hibernation) to reduce metabolic demands of maintaining high body temperature ( Tb). Torpor is typically characterized by coordinated declines in Tb and metabolic rate (MR) followed by active rewarming. Most hibernators experience periods of euthermy between bouts of torpor during which homeostatic processes are restored. In contrast, the common tenrec, a basoendothermic Afrotherian mammal, hibernates without interbout arousals and displays extreme flexibility in Tb and MR. We investigated the molecular basis of this plasticity in tenrecs by profiling the liver proteome of animals that were active or torpid with high and more stable Tb (∼32°C) or lower Tb (∼14°C). We identified 768 tenrec liver proteins, of which 50.9% were differentially abundant between torpid and active animals. Protein abundance was significantly more variable in active cold and torpid compared with active warm animals, suggesting poor control of proteostasis. Our data suggest that torpor in tenrecs may lead to mismatches in protein pools due to poor coordination of anabolic and catabolic processes. We propose that the evolution of endothermy leading to a more realized homeothermy of boreoeutherians likely led to greater coordination of homeostatic processes and reduced mismatches in thermal sensitivities of metabolic pathways. </jats:p

Elephant seal muscle cells adapt to sustained glucocorticoid exposure by shifting their metabolic phenotype

AbstractElephant seals experience natural periods of prolonged food deprivation while breeding, molting, and undergoing postnatal development. Prolonged food deprivation in elephant seals increases circulating glucocorticoids without inducing muscle atrophy, but the cellular mechanisms that allow elephant seals to cope with such conditions remain elusive. We generated a cellular model and conducted transcriptomic, metabolic, and morphological analyses to study how seal cells adapt to sustained glucocorticoid exposure. Seal muscle progenitor cells differentiate into contractile myotubes with a distinctive morphology, gene expression profile, and metabolic phenotype. Exposure to dexamethasone at three ascending concentrations for 48h modulated the expression of 6 clusters of genes related to structural constituents of muscle and pathways associated with energy metabolism and cell survival. Knockdown of the glucocorticoid receptor (GR) and downstream expression analyses corroborated that GR mediates the observed effects. Dexamethasone also decreased cellular respiration, shifted the metabolic phenotype towards glycolysis, and induced mitochondrial fission and dissociation of mitochondria-ER interactions without decreasing cell viability. Knockdown of DDIT4, a GR target involved in the dissociation of mitochondria-ER membranes, recovered respiration and modulated antioxidant gene expression. These results show that adaptation to sustained glucocorticoid exposure in elephant seal myotubes involves a metabolic shift toward glycolysis, which is supported by alterations in mitochondrial morphology and a reduction in mitochondria-ER interactions, resulting in decreased respiration without compromising cell survival.</jats:p