Seasonal variation in harbour seal (Phoca vitulina) blubber cortisol - A novel indicator of physiological state?

The authors would also like to thank DRG International Inc. for their financial support that enabled the attendance of The European Cetacean Society Conference in 2012 where part of this work was presented. This work was partly supported by the National Capability Funding from the Natural Environment Research Council and by a School of Biology Class Grant.Cortisol is one of the main glucocorticoid hormones involved in both the mammalian stress response, and in fat metabolism and energy regulation, making it of increasing interest as a biomarker for stress, health and overall physiological state. However, transient stress responses to animal handling and sampling may be important sources of measurement artefact when investigating circulating concentrations of this hormone in wildlife. Here, cortisol concentrations were measured in the plasma and, for the first time, in the blubber of live captured adult harbour seals (Phoca vitulina). Plasma cortisol concentrations were positively correlated with capture time, suggesting that they were largely driven by a stress response to the capture event. In contrast, blubber cortisol concentrations were shown not to be significantly affected by capture time and varied significantly by sex and by season, with higher concentrations during natural fasting periods of their life cycle, particularly during the moult. These results suggest that cortisol may play a key role in increased fat metabolism during highly energetically demanding periods, and that blubber concentrations have the potential to be used as physiological state indicators in phocid seals.Publisher PDFPeer reviewe

Designing a Tablet-based Intervention to Support Self-checking for Melanoma

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Determining pregnancy status in harbour seals using progesterone concentrations in blood and blubber

The authors would like to thank all the staff and students who helped collect the samples in the field, particularly Simon Moss, Matt Bivins, Paul Thompson, Isla Graham and Tim Cândido Barton. This study was made possible through funding from the UKRI Natural Environment Research Council (grant numbers SMRU10001 and NE/R015007/1), the Scottish Government (grant number MMSS/002/15), Beatrice Offshore Wind Ltd. (BOWL), Moray Offshore Renewables Ltd. (MORL), Marine Scotland Science, The Crown Estate and Highlands and Island Enterprise.Peer reviewedPostprintPostprin

Using blubber explants to investigate adipose function in grey seals:glycolytic, lipolytic and gene expression responses to glucose and hydrocortisone

Adipose tissue is fundamental to energy balance, which underpins fitness and survival. Knowledge of adipose regulation in animals that undergo rapid fat deposition and mobilisation aids understanding of their energetic responses to rapid environmental change. Tissue explants can be used to investigate adipose regulation in wildlife species with large fat reserves, when opportunities for organismal experimental work are limited. We investigated glucose removal, lactate, glycerol and NEFA accumulation in media, and metabolic gene expression in blubber explants from wild grey seals. Glycolysis was higher in explants incubated in 25 mM glucose (HG) for 24 h compared to controls (C: 5.5 mM glucose). Adipose-derived lactate likely contributes to high endogenous glucose production in seals. Lipolysis was not stimulated by HG or high hydrocortisone (HC: 500 nM hydrocortisone) and was lower in heavier animals. HC caused NEFA accumulation in media to decrease by ~30% relative to C in females, indicative of increased lipogenesis. Lipolysis was higher in males than females in C and HG conditions. Lower relative abundance of 11-β-hydroxysteroid dehydrogenase 1 mRNA in HG explants suggests glucose involvement in blubber cortisol sensitivity. Our findings can help predict energy balance responses to stress and nutritional state in seals, and highlight the use of explants to study fat tissue function in wildlife

Persistent organic pollutant burden, experimental POP exposure and tissue properties affect metabolic profiles of blubber from grey seal pups

Persistent organic pollutants (POPs) are toxic, ubiquitous, resist breakdown, bioaccumulate in living tissue and biomagnify in food webs. POPs can also alter energy balance in humans and wildlife. Marine mammals experience high POP concentrations, but consequences for their tissue metabolic characteristics are unknown. We used blubber explants from wild, grey seal (Halichoerus grypus) pups to examine impacts of intrinsic tissue POP burden and acute experimental POP exposure on adipose metabolic characteristics. Glucose use, lactate production and lipolytic rate differed between matched inner and outer blubber explants from the same individuals and between feeding and natural fasting. Glucose use decreased with blubber dioxin-like PCBs (DL-PCB) and increased with acute experimental POP exposure. Lactate production increased with DL-PCBs during feeding, but decreased with DL-PCBs during fasting. Lipolytic rate increased with blubber dichlorodiphenyltrichloroethane (DDT) and its metabolites (DDX) in fasting animals, but declined with DDX when animals were feeding. Our data show that POP burdens are high enough in seal pups to alter adipose function early in life, when fat deposition and mobilisation are vital. Such POP-induced alterations to adipose glucose use may significantly alter energy balance regulation in marine top predators with the potential for long term impacts on fitness and survival

Research in association with New Seal Licensing System : Research on the population structure of harbour seals

The population structure of harbour seals (Phoca vitulina) around Scotland was investigated using different genetic markers and approaches. This allowed discrete population units or metapopulations to be identified. The population genetic structure is compared to the recently defined harbour seal management regions (SCOS, 2011), ensuring Scottish Government’s regional management procedures and plans for harbour seals are based on genetic data as well as the currently employed ecological haulout and pupping site data. Analysis of DNA samples from a total of 453 individuals around Scotland including samples from comparative regions in the UK and Europe (including an out-group of Pacific harbour seals) was carried out. Following some initial trials the most appropriate population differentiation analysis comprised 10 putative populations across all the samples analysed. Focusing on Scotland, Bayesian clustering analysis clearly separated Scotland from England, France and the Dutch Wadden Sea. In this scenario 3 clusters were generally identified: a) Norway, b)West Coast of Scotland/Northern Ireland and c) Pentland Firth / Orkney / Shetland / Moray Firth / Tay and Eden with some degree of shared individuals between them. Examining the Scottish populations alone indicated there might be some additional separation between the Tay and Eden compared to the other north and east coast groups. Within the Scottish populations a number of harbour seal Management Areas have been assigned based on haul outs and breeding sites (SCOS, 2011). The result of the genetic analyses reported here clearly supports the designation and definition of these Areas. Allelic diversity and heterozygosity are standard measures that assess the level of inbreeding which populations display as a reflection of their ‘genetic health’. The populations with relatively good sample sizes and low levels of genetic diversity were Shetland (n=2.545, HO=0.363) and the Outer Hebrides (2.467, HO= 0.331). It has been widely shown that inbreeding, translated as very low levels of genetic diversity in wild populations is correlated with disease such as cancer (Acevedo-Whitehouse et al. 2003) and with susceptibility to pathogens such as parasites (Rijks et al. 2008) among others.Publisher PDFPublisher PD

Proteome profiling reveals opportunities to investigate biomarkers of oxidative stress and immune responses in blubber biopsies from free-ranging baleen whales

Funding: The authors would like to thank the Strategic Environmental Research and Development Program (SERDP) who supported parts of this work (grant numbers RC-2113 and RC-2337). The authors would also like to thank the National Environment Research Council (NE/R015007/1) for their support for parts of this work.Over 25% of cetacean species worldwide are listed as critically endangered, endangered or vulnerable by the International Union for Conservation of Nature. Objective and widely applicable tools to assess cetacean health are therefore vital for population monitoring and to inform conservation initiatives. Novel blubber biomarkers of physiological state are examples of such tools that could be used to assess overall health. Proteins extracted from blubber likely originate from both the circulation and various cell types within the tissue itself, and their expression is responsive to signals originating from other organs and the nervous system. Blubber proteins can therefore capture information on physiological stressors experienced by individuals at the time of sampling. For the first time, we assess the feasibility of applying shotgun proteomics to blubber biopsy samples collected from free-ranging baleen whales. Samples were collected from minke whales (Balaenoptera acutorostrata) (n = 10) in the Gulf of St Lawrence, Canada. Total protein was extracted using a RIPA cell lysis and extraction buffer-based protocol. Extracted proteins were separated and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry. We mapped proteins to known biological pathways and determined whether they were significantly enriched based on the proteome profile. A pathway enrichment map was created to visualize overlap in tissue-level biological processes. Amongst the most significantly enriched biological pathways were those involved in immune system function: inflammatory responses, leukocyte-mediated immunity and the humoral immune response. Pathways associated with responses to oxidative stress were also enriched. Using a suite of such protein biomarkers has the potential to better assess the overall health and physiological state of live individuals through remote biopsy sampling. This information is vital for population health assessments to predict population trajectories, and ultimately guide and monitor conservation priorities and initiatives.Peer reviewe

Not just fat : investigating the proteome of cetacean blubber tissue

Mammalian adipose tissue is increasingly being recognized as an endocrine organ involved in the regulation of a number of metabolic processes and pathways. It responds to signals from different hormone systems and the central nervous system, and expresses a variety of protein factors with important paracrine and endocrine functions. This study presents a first step towards the systematic analysis of the protein content of cetacean adipose tissue, the blubber, in order to investigate the kinds of proteins present and their relative abundance. Full depth blubber subsamples were collected from dead-stranded harbour porpoises (Phocoena phocoena) (n = 21). Three total protein extraction methods were trialled, and the highest total protein yields with the lowest extraction variability were achieved using a RIPA cell lysis and extraction buffer based protocol. Extracted proteins were separated using 1D Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and identified using nanoflow Liquid Chromatography Electrospray Ionization in tandem with Mass Spectrometry (nLC-ESI–MS/MS). A range of proteins were identified (n = 295) and classed into eight functional groups, the most abundant of which were involved in cell function and metabolism (45%), immune response and inflammation (15%) and lipid metabolism (11%). These proteins likely originate both from the various cell types within the blubber tissue itself, and from the circulation. They therefore have the potential to capture information on the cellular and physiological stresses experienced by individuals at the time of sampling. The importance of this proteomic approach is two-fold: Firstly, it could help to assign novel functions to marine mammal blubber in keeping with current understanding of the multi-functional role of adipose tissue in other mammals. Secondly, it could lead to the development of a suite of biomarkers to better monitor the physiological state and health of live individuals though remote blubber biopsy sampling.Publisher PDFPeer reviewe

Consequences of <i>in vitro</i> benzyl butyl phthalate exposure for blubber gene expression and insulin-induced Akt activation in juvenile grey seals

Plastic and plasticiser pollution of marine environments is a growing concern. Although phthalates, one group of plasticisers, are rapidly metabolised by mammals, they are found ubiquitously in humans and have been linked with metabolic disorders and altered adipose function. Phthalates may also present a threat to marine mammals, which need to rapidly accumulate and mobilise their large fat depots. High molecular weight (HMW) phthalates may be most problematic because they can accumulate in adipose. We used blubber explants from juvenile grey seals to examine the effects of overnight exposure to the HMW, adipogenic phthalate, benzyl butyl phthalate (BBzP) on expression of key adipose-specific genes and on phosphorylation of Akt in response to insulin. We found substantial differences in transcript abundance of Pparγ, Insig2, Fasn, Scd, Adipoq and Lep between moult stages, when animals were also experiencing differing mass changes, and between tissue depths, which likely reflect differences in blubber function. Akt abundance was higher in inner compared to outer blubber, consistent with greater metabolic activity in adipose closer to muscle than skin, and its phosphorylation was stimulated by insulin. Transcript abundance of Pparγ and Fasn (and Adipoq in some animals) were increased by short term (30 min) insulin exposure. In addition, overnight in vitro BBzP exposure altered insulin-induced changes in Pparγ (and Adipoq in some animals) transcript abundance, in a tissue depth and moult stage-specific manner. Basal or insulin-induced Akt phosphorylation was not changed. BBzP thus acted rapidly on the transcript abundance of key adipose genes in an Akt-independent manner. Our data suggest phthalate exposure could alter seal blubber development or function, although the whole animal consequences of these changes are not yet understood. Knowledge of typical phthalate exposures and toxicokinetics would help to contextualise these findings in terms of phthalate-induced metabolic disruption risk and consequences for marine mammal health

Obtaining accurate glucose measurements from wild animals under field conditions:comparing a hand held glucometer with a standard laboratory technique in grey seals

Glucose is an important metabolic fuel and circulating levels are tightly regulated in most mammals, but can drop when body fuel reserves become critically low. Glucose is mobilized rapidly from liver and muscle during stress in response to increased circulating cortisol. Blood glucose levels can thus be of value in conservation as an indicator of nutritional status and may be a useful, rapid assessment marker for acute or chronic stress. However, seals show unusual glucose regulation: circulating levels are high and insulin sensitivity is limited. Accurate blood glucose measurement is therefore vital to enable meaningful health and physiological assessments in captive, wild or rehabilitated seals and to explore its utility as a marker of conservation relevance in these animals. Point-of-care devices are simple, portable, relatively cheap and use less blood compared with traditional sampling approaches, making them useful in conservation-related monitoring. We investigated the accuracy of a hand-held glucometer for ‘instant’ field measurement of blood glucose, compared with blood drawing followed by laboratory testing, in wild grey seals (Halichoerus grypus), a species used as an indicator for Good Environmental Status in European waters. The glucometer showed high precision, but low accuracy, relative to laboratory measurements, and was least accurate at extreme values. It did not provide a reliable alternative to plasma analysis. Poor correlation between methods may be due to suboptimal field conditions, greater and more variable haematocrit, faster erythrocyte settling rate and/or lipaemia in seals. Glucometers must therefore be rigorously tested before use in new species and demographic groups. Sampling, processing and glucose determination methods have major implications for conclusions regarding glucose regulation, and health assessment in seals generally, which is important in species of conservation concern and in development of circulating glucose as a marker of stress or nutritional state for use in management and monitoring