Functional Attenuation of UCP1 as the Potential Mechanism for a Thickened Blubber Layer in Cetaceans.

Uncoupling protein 1 (UCP1) is an essential protein in the mitochondrial inner membrane that mediates nonshivering thermogenesis (NST) and plays an important role in thermoregulation and fat deposition. However, the relationship between the evolution of UCP1 and fat deposition in the blubber layer in cetaceans remains unclear. Here, frameshift mutations, premature termination, and relaxed selection pressure (ω = 0.9557, P < 0.05) were detected in UCP1 in cetaceans, suggesting that UCP1 was inactivated during cetacean evolution. By time estimation, it was found that the inactivation of UCP1 in cetaceans occurred between 53.1 and 50.2 Ma. However, combined with findings from immunohistochemical analysis of the blubber layer of the Yangtze finless porpoise and in vitro functional assays, a premature termination of cetacean UCP1 resulted in a reduction of UCP1-mediated NST capacity (about 50%) and lipolytic capacity (about 40%), both of which were beneficial to maintain blubber layer and body temperature without excessive fat consumption. This study provides new insights into the molecular mechanisms of the blubber thickening in cetaceans and highlights the importance of UCP1 attenuation in cetaceans for secondary aquatic adaptation.

Effects of cortisol, epinephrine, and bisphenol contaminants on the transcriptional landscape of marine mammal blubber.

Top ocean predators such as marine mammals are threatened by intensifying anthropogenic activity, and understanding the combined effects of multiple stressors on their physiology is critical for conservation efforts. We investigated potential interactions between stress hormones and bisphenol contaminants in a model marine mammal, the northern elephant seal (NES). We exposed precision-cut adipose tissue slices (PCATS) from blubber of weaned NES pups to cortisol (CORT), epinephrine (EPI), bisphenol A (BPA), bisphenol S (BPS), or their combinations (CORT-EPI, BPA-EPI, and BPS-EPI) ex vivo and identified hundreds of genes that were differentially regulated in response to these treatments. CORT altered expression of genes associated with lipolysis and adipogenesis, whereas EPI and CORT-EPI-regulated genes were associated with responses to hormones, lipid and protein turnover, immune function, and transcriptional and epigenetic regulation of gene expression, suggesting that EPI has wide-ranging and prolonged impacts on the transcriptional landscape and function of blubber. Bisphenol treatments alone had a weak impact on gene expression compared with stress hormones. However, the combination of EPI with bisphenols altered expression of genes associated with inflammation, cell stress, DNA damage, regulation of nuclear hormone receptor activity, cell cycle, mitochondrial function, primary ciliogenesis, and lipid metabolism in blubber. Our results suggest that CORT, EPI, bisphenols, and their combinations impact cellular, immune, and metabolic homeostasis in marine mammal blubber, which may affect the ability of marine mammals to sustain prolonged fasting during reproduction and migration, renew tissues, and mount appropriate responses to immune challenges and additional stressors.

Quantitative fatty acid signature analysis reveals a high level of dietary specialization in killer whales across the North Atlantic.

Quantifying the diet composition of apex marine predators such as killer whales (Orcinus orca) is critical to assessing their food web impacts. Yet, with few exceptions, the feeding ecology of these apex predators remains poorly understood. Here, we use our newly validated quantitative fatty acid signature analysis (QFASA) approach on nearly 200 killer whales and over 900 potential prey to model their diets across the 5000 km span of the North Atlantic. Diet estimates show that killer whales mainly consume other whales in the western North Atlantic (Canadian Arctic, Eastern Canada), seals in the mid-North Atlantic (Greenland), and fish in the eastern North Atlantic (Iceland, Faroe Islands, Norway). Nonetheless, diet estimates also varied widely among individuals within most regions. This level of inter-individual feeding variation should be considered for future ecological studies focusing on killer whales in the North Atlantic and other oceans. These estimates reveal remarkable population- and individual-level variation in the trophic ecology of these killer whales, which can help to assess how their predation impacts community and ecosystem dynamics in changing North Atlantic marine ecosystems. This new approach provides researchers with an invaluable tool to study the feeding ecology of oceanic top predators.

Connaître en détails la composition du régime alimentaire des grands prédateurs marins tels que les orques (Orcinus orca) est primordial afin d'évaluer leurs impacts sur les écosystèmes. Pourtant, à quelques exceptions près, l'écologie alimentaire de ces super-prédateurs reste mal comprise. Ici, nous utilisons notre nouvelle approche d'analyse quantitative des signatures d'acides gras (QFASA) sur près de 200 orques et plus de 900 proies potentielles pour modéliser leur régime alimentaire à travers l'Atlantique Nord. Les estimations de leurs régimes alimentaires montrent que les orques consomment principalement d'autres baleines dans l'ouest de l'Atlantique Nord (Arctique canadien, Est du Canada), des phoques dans le milieu de l'Atlantique Nord (Groenland) et des poissons dans l'est de l'Atlantique Nord (Islande, îles Féroé, Norvège). Néanmoins, ces estimations varient considérablement d'un individu à l'autre dans la plupart des régions. Cette variation alimentaire importante entre les individus doit être prise en compte dans les futures études écologiques qui s'intéressent aux orques de l'Atlantique Nord et d'ailleurs. Ces estimations révèlent des variations remarquables dans l'écologie trophique des orques tant au niveau des population que de l'individu, ce qui peut aider à évaluer l'impact de leur prédation sur la dynamique des communautés et des écosystèmes dans un contexte de changements climatiques en l'Atlantique Nord. Cette nouvelle approche fournit aux chercheurs un outil inestimable pour étudier l'écologie alimentaire des super-prédateurs océaniques.

Varying Diet Composition Causes Striking Differences in Legacy and Emerging Contaminant Concentrations in Killer Whales across the North Atlantic.

Lipophilic persistent organic pollutants (POPs) tend to biomagnify in food chains, resulting in higher concentrations in species such as killer whales (Orcinus orca) feeding on marine mammals compared to those consuming fish. Advancements in dietary studies include the use of quantitative fatty acid signature analysis (QFASA) and differentiation of feeding habits within and between populations of North Atlantic (NA) killer whales. This comprehensive study assessed the concentrations of legacy and emerging POPs in 162 killer whales from across the NA. We report significantly higher mean levels of polychlorinated biphenyls (PCBs), organochlorine pesticides, and flame retardants in Western NA killer whales compared to those of Eastern NA conspecifics. Mean ∑PCBs ranged from ∼100 mg/kg lipid weight (lw) in the Western NA (Canadian Arctic, Eastern Canada) to ∼50 mg/kg lw in the mid-NA (Greenland, Iceland) to ∼10 mg/kg lw in the Eastern NA (Norway, Faroe Islands). The observed variations in contaminant levels were strongly correlated with diet composition across locations (inferred from QFASA), emphasizing that diet and not environmental variation in contaminant concentrations among locations is crucial in assessing contaminant-associated health risks in killer whales. These findings highlight the urgency for implementing enhanced measures to safely dispose of POP-contaminated waste, prevent further environmental contamination, and mitigate the release of newer and potentially harmful contaminants.

Changes in apolipoprotein abundance dominate proteome responses to prolonged fasting in elephant seals.

Unlike many animals that reduce activity during fasting, northern elephant seals (NES) undergo prolonged fasting during energy-intensive life-history stages such as reproduction and molting, fueling fasting energy needs by mobilizing fat stores accrued during foraging. NES display several unique metabolic features such as high fasting metabolic rates, elevated blood lipid and high-density lipoprotein (HDL) cholesterol levels, efficient protein sparing and resistance to oxidative stress during fasting. However, the cellular mechanisms that regulate these adaptations are still not fully understood. To examine how metabolic coordination is achieved during prolonged fasting, we profiled changes in blubber, skeletal muscle and plasma proteomes of adult female NES over a 5 week fast associated with molting. We found that while blubber and muscle proteomes were remarkably stable over fasting, over 50 proteins changed in abundance in plasma, including those associated with lipid storage, mobilization, oxidation and transport. Apolipoproteins dominated the blubber, plasma and muscle proteome responses to fasting. APOA4, APOE and APOC3, which are associated with lipogenesis and triglyceride accumulation, decreased, while APOA1, APOA2 and APOM, which are associated with lipid mobilization and HDL function, increased over fasting. Our findings suggest that changes in apolipoprotein composition may underlie the maintenance of high HDL levels and, together with adipokines and hepatokines that facilitate lipid catabolism, may mediate the metabolic transitions between feeding and fasting in NES. Many of these proteins have not been previously studied in this species and provide intriguing hypotheses about metabolic regulation during prolonged fasting in mammals.

Blubber Cortisol-Based Approach to Explore the Endocrine Responses of Indo-Pacific Humpback Dolphins (Sousa chinensis) to Diet Shifts and Contaminant Exposure.

The detrimental effects of contaminant exposure and changes in the availability of food resources are still of concern for Indo-Pacific humpback dolphins (Sousa chinensis) of the Pearl River Estuary (PRE). Here, we validated and applied a blubber cortisol biomarker approach to assess the physiological responses of PRE dolphins to various pollutants and diet changes during 2008-2018 (n = 70). For calves, generalized additive models (GAMs) revealed that cortisol levels varied significantly by month and were positively correlated with the body length, owing to significant maternal transfer of hormones. The significantly positive correlation between length-adjusted cortisol levels in calf and the annual calf mortality ratios suggested that during years of high calf mortality, these animals were highly stressed before they die. For noncalves, blubber cortisol levels in diseased animals were significantly higher than those in "healthy" ones. Chromium (Cr) and dichlorodiphenyltrichloroethanes displayed a significant and positive relationship with blubber cortisol levels, suggesting that contaminant-mediated endocrine disruption effects may have occurred in noncalves. The GAMs indicated a decreasing trend of noncalf's blubber cortisol levels over an 11-year span, which can be explained by their declining contaminant accumulation levels due to a significant dietary shift from eating highly contaminated fishes to less polluted ones.

Blubber and serum cortisol concentrations as indicators of the stress response and overall health status in striped dolphins.

The impacts of environmental changes and anthropogenic threats in marine mammals are a growing concern for their conservation. In recent years, efforts have been directed to understand how marine mammals cope with stressors and to assess and validate stress biomarkers, mainly levels of glucocorticoid hormones (e.g. cortisol) in certain body tissues. The aims of this study were to assess the impact of different causes of stranding (chronically affected and bycaught striped dolphins) on cortisol concentrations in serum and in blubber; and to evaluate the association between cortisol levels in these tissues. Blubber and blood samples were collected from striped dolphins (n = 42) stranded on the Mediterranean coast between 2012 and 2018. Cortisol concentrations were measured by using enzyme immunoassay. A high correlation was found between circulating and blubber cortisol concentrations (R2 = 0.85, p < 0.01). Necropsies and pathological studies concluded that a third of the dolphins were bycaught in fishing nets and released by fishermen (Bycaught animals group), while the other two thirds were euthanized, or died, due to a disease or chronic condition (e.g. calves separated from the mother or animals infected with dolphin morbillivirus or Brucella ceti) that impeded survival (Chronically affected animals group). Cortisol concentrations (mean ± SD) were six times higher in chronically affected animals (35.3 ± 23 ng cortisol/g blubber and 6.63 ± 3.22 µg cortisol/dl serum) compared to those bycaught in fishing nets (6.2 ± 4.3 ng cortisol/g blubber and 1.15 ± 1.51 µg cortisol/dl serum). Results suggests that serum and blubber cortisol concentrations can contribute in inferring the overall health and welfare of free-ranging cetaceans. However, further research is required to understand better the kinetics of blubber cortisol incorporation and removal, the factors involved in these processes, and the local conversion of cortisol in the blubber.

Complementary use of stable isotopes and fatty acids for quantitative diet estimation of sympatric predators, the Antarctic pack-ice seals.

The quantitative use of stable isotopes (SIs) for trophic studies has seen a rapid growth whereas fatty acid (FA) studies remain mostly qualitative. We apply the Bayesian tool MixSIAR to both SI and FA data to estimate the diet of three sympatric predators: the crabeater (Lobodon carcinophaga), Weddell (Leptonychotes weddellii) and leopard seal (Hydrurga leptonyx). We used SI data of their vibrissae and FA data of their outer blubber to produce comparable diet estimates for the same individuals. Both SI and FA models predicted the same main diet components, although the predicted proportions differed. For the crabeater seal, both methods identified krill, Euphausia superba, as the main, and almost exclusive, food item, although the FA model estimated a slightly lower proportion, potentially due to the low lipid content of krill compared to the fish species used in the model. For the Weddell seal the FA model identified the fish Pleuragramma antarcticum as the most important prey, whereas the SI model was not able to distinguish among prey species, identifying a 'fish-squid' group as the main diet component. For the leopard seal, both models identified krill as the main contributor; however, the predicted proportions for the secondary sources differed. Although vibrissae and outer blubber may not represent the same timeframe, the use of MixSIAR with FA data provides diet estimates comparable to those obtained with SI data, thus, both approaches were complimentary. The use of both biotracers offers a feasible option to study diets of wild animals in a quantitative manner.

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

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.

The influence of physiological status on the reproductive behaviour of humpback whales (Megaptera novaeangliae).

For most cetacean species, there is little known about how an individual's physiology influences its behaviour. Humpback whales (Megaptera novaeangliae) are a good candidate to examine such links as they have a well-described distribution and behaviour, can be consistently sampled using remote biopsy systems, and have been the subject of several previous endocrine studies. The objective here was to examine whether a female humpback whale's social state (i.e. escorted by a male or not) is related to her endocrine condition, and whether male dominance ranking is related to testosterone levels. Skin and blubber biopsies were collected from the east and west Australian humpback whale populations in 2010-2016 (n = 252) at multiple times throughout the winter-spring breeding season. Steroid hormones were extracted from blubber and concentrations of progesterone (a marker for pregnancy), testosterone (a marker of male testicular activity) and oestradiol (a potential marker of ovarian activity) measured using enzyme-immunoassays. Principal escorts-the dominant males in mixed sex groups-had significantly higher blubber testosterone levels (mean ±â€¯SE; 1.43 ±â€¯0.20 ng/g wet weight) than subordinate, secondary escorts (0.69 ±â€¯0.06 ng/g wet weight). Females that were escorted by males typically possessed elevated blubber oestradiol levels (1.96 ±â€¯0.25 ng/g wet weight; p = 0.014); few were considered to be pregnant (p = 0.083). 'Unescorted' females displayed characteristically lower blubber oestradiol levels (0.56 ±â€¯0.06 ng/g wet weight). Together, these results are consistent with 'challenge hypothesis' theory and suggest the existence of associated reproductive patterns in humpback whales.

Blubber cortisol levels in humpback whales (Megaptera novaeangliae): A measure of physiological stress without effects from sampling.

Baleen whales are vulnerable to environmental impacts due to low fecundity, capital breeding strategies, and their reliance on a large amount of prey resources over large spatial scales. There has been growing interest in monitoring health and physiological stress in these species but, to date, few measures have been validated. The purpose of this study was to examine whether blubber cortisol could be used as a measure of physiological stress in humpback whales. Cortisol concentrations were initially compared between live, presumably 'healthy' whales (n = 187) and deceased whales (n = 35), which had died after stranding or entanglement, or washed ashore as a carcass. Deceased whales were found to have significantly higher cortisol levels (mean ± SD; 5.47 ± 4.52 ng/g) than live whales (0.51 ± 0.14 ng/g; p < 0.001), particularly for those animals that had experienced prolonged trauma (e.g. stranding) prior to death. Blubber cortisol levels in live whales were then examined for evidence of life history-related, seasonal, or sampling-related effects. Life history group and sampling-related factors, such as encounter time and the number of biopsy sampling attempts per animal, were found to be poor predictors of blubber cortisol levels in live whales. In contrast, blubber cortisol levels varied seasonally, with whales migrating north towards the breeding grounds in winter having significantly higher levels (0.54 ± 0.21 ng/g, p = 0.016) than those migrating south towards the feeding grounds in spring (0.48 ± 1.23 ng/g). These differences could be due to additional socio-physiological stress experienced by whales during peaks in breeding activity. Overall, blubber cortisol appears to be a suitable measure of chronic physiological stress in humpback whales.

Blubber steroid hormone profiles as indicators of physiological state in free-ranging common bottlenose dolphins (Tursiops truncatus).

Blubber has been proposed as a possible alternative to blood in the assessment of endocrine physiology in marine mammals because it can be collected via remote biopsy, which removes some of the confounding variables and logistical constraints associated with blood collection. To date, few studies have directly assessed the relationships between circulating versus blubber steroid hormone profiles in marine mammals, and these studies have been limited to a small subset of steroid hormones, which collectively limit the current utility of blubber steroid hormone measurements. In this study, we used liquid-chromatography tandem-mass spectrometry (LC-MS/MS) to screen for 16 steroid hormones in matched blood and blubber samples from free-ranging common bottlenose dolphins (Tursiops truncatus). Seven steroid hormones were detected and quantified, including two progestogens, two androgens, and three corticosteroids. Using principal components analysis (PCA), we explored relationships between hormones in both matrices and three physiological states: sexual maturity, pregnancy, and acute stress response. Plasma and blubber testosterone and its precursors, 17-hydroxyprogesterone and androstenedione, loaded to the first principal component (PC1), and PC1 scores were higher in mature males. Plasma and blubber progesterone loaded to PC2, and pregnant/probable pregnant females had significantly higher PC2 scores. Pregnant females also had higher PC1 scores than other females, suggesting differences in androgen profiles between these groups. There was disagreement between plasma and blubber corticosteroid profiles, as indicated by their loading to different PCs; plasma corticosteroids loaded to PC3 and blubber corticosteroids to PC4. PC3 scores were significantly predicted by elapsed time to blood collection (i.e., time between initiating the capture process and blood collection), while elapsed time to blubber collection significantly predicted PC4 scores, indicating that corticosteroid profiles shift in both tissues during acute stress. Corticosteroid profiles were not related to demographic group, site-month, body mass index, water temperature, or time spent outside of the water on the processing boat. Overall, these results demonstrate that blubber steroid hormone profiles reflect changes in endocrine function that occur over broad temporal scales.

From low to high latitudes: changes in fatty acid desaturation in mammalian fat tissue suggest a thermoregulatory role.

BACKGROUND: Most fatty acids (FAs) making up the adipose tissue in mammals have a dietary origin and suffer little modification when they are stored. However, we propose that some of those FAs, specifically those that can be synthesised or modified by mammals, are also being influenced by thermal forces and used as part of the mechanism to regulate core body temperature. As FA desaturation increases, adipose tissues can reach colder temperatures without solidifying. The ability to cool the superficial fat tissues helps create a thermal gradient, which contributes to body heat loss reduction. Therefore, it is expected that animals exposed to colder environments will possess adipose tissues with higher proportions of desaturated FAs. Here, through a model selection approach that accounts for phylogeny, we investigate how the variation in FA desaturation in 54 mammalian species relates to the thermal proxies: latitude, physical environment (terrestrial, semi-aquatic and fully-aquatic) and hair density. RESULTS: The interaction between the environment (terrestrial, semi- or fully-aquatic) and the latitude in which the animals lived explained best the variation of FA desaturation in mammals. Aquatic mammals had higher FA desaturation compared to terrestrial mammals. Semi-aquatic mammals had significantly higher levels of desaturated FAs when living in higher latitudes whereas terrestrial and fully-aquatic mammals did not. To account for dietary influence, a double bond index was calculated including all FAs, and revealed no correlation with latitude in any of the groups. CONCLUSIONS: We propose that FA modification is an important component of the thermoregulatory strategy, particularly in semi-aquatic mammals. Potentially this is because, like terrestrial mammals, they experience the greatest air temperature variations across latitudes, but they lack a thick fur coat and rely primarily on their blubber. Unlike fully-aquatic mammals, extremely thick blubber is not ideal for semi-aquatic mammals, as this is detrimental to their manoeuvrability on land. Therefore, the adipose tissue in semi-aquatic mammals plays a more important role in keeping warm, and the modification of FAs becomes crucial to withstand cold temperatures and maintain a pliable blubber.

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.

Evaluation of respiratory vapour and blubber samples for use in endocrine assessments of bottlenose dolphins (Tursiops spp.).

Blubber and respiratory vapour ('blow') are now commonly used for endocrine studies on cetaceans, primarily because they can be obtained using minimally invasive methods. For many species, these samples have yet to be validated for these purposes. The objective of this study was to examine the performance of blow and blubber hormone monitoring, relative to serum hormone monitoring, for evaluating the reproductive and adrenal condition of captive bottlenose dolphins (Tursiops spp.). Eighteen bottlenose dolphins were sampled five times for serum and blow and twice for blubber throughout a one-year period. Concentrations of progesterone, testosterone, oestradiol and cortisol were measured in each sample type. Hormone levels were examined in relation to dolphin age, sex, reproductive status, season, time of sample collection (morning/afternoon) and collection type (in- or out-of-water sampling). Patterns in hormone levels were similar for serum and blubber. For instance, in both sample types, progesterone levels were significantly higher in pregnant (serum: 34.10 ±â€¯8.64 ng/mL; blubber: 13.01 ±â€¯0.72 ng/g) than in non-pregnant females (serum: 0.32 ±â€¯0.09 ng/mL; blubber: 1.17 ±â€¯0.10 ng/g). This pattern was not detected in blow, primarily because seawater contamination, nylon sampling materials and variable sample volumes influenced measured concentrations. In addition, the respiratory water content of a blow sample is known to affect measured hormone levels. Two methods were trialled to control for variability in sample volumes and dilution: (1) normalising blow hormone concentrations relative to urea nitrogen levels (a potential endogenous standard), and (2) measuring the relative proportions (i.e. ratios) of blow hormones. These correction measures had little influence on blow hormone results. Further refinement of blow hormone monitoring methods is required before they can be used for reproductive or adrenal assessments of bottlenose dolphins. Blubber, on the other hand, should be a suitable proxy for serum when attempting to classify pregnancy status and male maturity in these species.

Remote blubber sampling paired with liquid chromatography tandem mass spectrometry for steroidal endocrinology in free-ranging bottlenose dolphins (Tursiops truncatus).

Liquid chromatography tandem mass spectrometry allows for the measurement of steroid hormone suites in the blubber of marine mammals. By combining this technology with minimally invasive techniques such as remote biopsy, endocrine profiles can be assessed, allowing for studies of hormonal profile variation over time. In this study, we explored associations among different steroidogenic pathways and seasonal differences in blubber hormone profiles of free-ranging common bottlenose dolphins along the coast of South Carolina, USA. Male dolphins experience a peak in testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone in the spring, likely related to an upregulation of the androgen steroidogenic pathway during mating season. We also observed increased cortisol concentrations during summer compared to winter. Among females, there was an increase in androstenedione with elevated progesterone concentrations indicative of pregnancy, highlighting another potential endocrine marker for pregnancy in free-ranging dolphins. This work emphasizes the importance of selecting the appropriate season for studies on endocrine status to effectively uncover physiological variation or disruption in free-ranging cetaceans.

Function and evolution of specialized endogenous lipids in toothed whales.

The Odontocetes (toothed whales) possess two types of specialized fat and, therefore, represent an interesting group when considering the evolution and function of adipose tissue. All whales have a layer of superficial blubber, which insulates and streamlines, provides buoyancy and acts as an energy reserve. Some toothed whales deposit large amounts of wax esters, rather than triacylglycerols, in blubber, which is unusual. Waxes have very different physical and physiological properties, which may impact blubber function. The cranial acoustic fat depots serve to focus sound during echolocation and hearing. The acoustic fats have unique morphologies; however, they are even more specialized biochemically because they are composed of a mix of endogenous waxes and triacylglycerols with unusual branched elements (derived from amino acids) that are not present in other mammals. Both waxes and branched elements alter how sound travels through a fat body; they are arranged in a 3D topographical pattern to focus sound. Furthermore, the specific branched-chain acid/alcohol synthesis mechanisms and products vary phylogenetically (e.g. dolphins synthesize lipids from leucine whereas beaked whales use valine). I propose that these specialized lipids evolved first in the head: wax synthesis first emerged to serve an acoustic function in toothed whales, with branched-chain synthesis adding additional acoustic focusing power, and some species secondarily retained wax synthesis pathways for blubber. Further research is necessary to elucidate specific molecular mechanisms controlling the synthesis and deposition of wax esters and branched-chain fatty acids, as well as their spatial deposition within tissues and within adipocytes.

Crying a river: how much salt-laden jelly can a leatherback turtle really eat?

Leatherback turtles (Dermochelys coriacea) are capital breeders that accumulate blubber (33 kJ g-1 wet mass) by hyperphagia on a gelatinous diet at high latitudes; they breed in the tropics. A jellyfish diet is energy poor (0.1-0.2 kJ g-1 wet mass) so leatherbacks must ingest large quantities. Two published estimates of feeding rate [50% body mass day-1 (on Rhizostoma pulmo) and 73% body mass day-1 (on Cyanea capillata)] have been criticised as too high. Jellyfish have high salt and water contents that must be removed to access organic material and energy. Most salt is removed (as NaCl) by paired lachrymal salt glands. Divalent ions are lost via the gut. In this study, the size of adult salt glands (0.622 kg for a 450 kg turtle; relatively three times the size of salt glands in cheloniid turtles) was measured for the first time by computed tomography scanning. Various published values for leatherback field metabolic rate, body fluid composition and likely blubber accumulation rates are combined with known jellyfish salt, water and organic compositions to calculate feasible salt gland secretion rates and feeding rates. The results indicate that leatherbacks can produce about 10-15 ml secretion g-1 salt gland mass h-1 (tear osmolality 1800 mOsm kg-1). This will permit consumption of 80% body mass day-1 of Ccapillata Calculations suggest that leatherbacks will find it difficult/impossible to accumulate sufficient blubber for reproduction in a single feeding season. Rapid jellyfish digestion and short gut transit times are essential.

Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4 g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations. Graphical abstract The steroid hormone profile is quantifiable from a single sample of bottlenose dolphin blubber using liquid chromatography tandem mass spectrometry. This profile can be applied to remotely collected dart biopsies and be used to determine reproductive or stress status of a wild-living dolphin.

Initial validation of blubber cortisol and progesterone as indicators of stress response and maturity in an otariid; the California sea lion (Zalophus californianus).

Chronic stress can have detrimental effects on an individual's health and reproductive success. The use of cortisol quantification as an indicator of stress in free-ranging cetaceans and phocids is increasing but no studies have applied this technique on blubber in otariids. We measured cortisol concentrations in blubber samples obtained from California sea lions, Zalophus californianus, stranded in San Diego County and those incidentally killed in the California drift gillnet fishery. We also measured progesterone concentrations to assess female reproductive status and, in males, as a potential secondary measure of adrenal steroid production. Blubber cortisol and progesterone values were compared across demographic groups (sex and maturity), season, and proportion blubber lipid extracted. Stranded animals (247.3±70.767SEng/gblubber) had significantly higher cortisol concentrations compared to fishery bycaught (8.1±2.108SEng/gblubber) animals. These findings are likely driven by inherent differences in the cause of death and associated nutritional state coupled with the mean duration of expiration for these two groups of animals (i.e., the duration from an animal's initial perception of the threat-to-self until death). The duration of transition from healthy state to death in stranded animals is on the order of many hours to weeks while in fishery bycaught animals, this transition occurs much more rapidly (i.e., seconds to tens of minutes). The presumed longer duration of the mortality event in stranded animals gives sufficient time for elevated cortisol to diffuse into the blubber. No significant differences between demographic groups, or season were found. However, blubber cortisol declined inversely with proportion blubber lipid extracted, suggesting utility in assessing long-term nutritional status. Blubber progesterone was significantly higher in mature females than immature females (153.8±54.546SEng/gblubber and 9.7±3.60SEng/gblubber respectively), containing on average 15 times more progesterone, irrespective of pregnancy state. Additionally, a significant relationship between mean cortisol and progesterone was found in males with >35% blubber lipid (p<0.0001). This study is an initial step in validating blubber cortisol and progesterone concentrations as a potential marker of stress response and reproductive state, respectively, in otariids. Especially when paired with dart biopsying, this approach could represent a relatively rapid way to assess baseline stress, nutritional status and reproductive states in otariids while minimizing the effects of sampling.