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

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

Exposure of harbour seals (Phoca vitulina) to Brucella in declining populations across Scotland

The authors acknowledge the NERC National Capability Funding grant number SMRU 10001 for the fundingSince 2000 there have been major declines in the abundance of Scottish harbour seals (Phoca vitulina). The causes of the declines remain uncertain. The aim of this study was to establish the extent to which the seals in the regions of greatest decline have been exposed to Brucella, a bacterial pathogen that causes reproductive failure in terrestrial mammalian hosts. Tissues from dead seals collected between 1992 and 2013 were cultured for Brucella (n=150). Serum samples collected from live capture-released seals (n=343) between 1997 and 2012 were tested for Brucella antibodies using the Rose Bengal plate agglutination test (RBT) and a competitive Enzyme-Linked Immunosorbent Assay (ELISA). 16% of seals cultured had Brucella isolated from one or more tissues but there were no pathological signs of infection. The cELISA results were more sensitive than the RBT results showing that overall, 25.4% of seals were seropositive with the highest seroprevalence in juveniles. As there was no evidence of either a higher seroprevalence, or higher circulating antibody levels in seropositive animals in the areas with the greatest declines, it was concluded that Brucella infection is likely not a major contributing factor to recent declines. However, the consistently high proportion of seals exposed to Brucella indicates possible endemicity in these populations, likely due to B. pinnipedialis, which has demonstrated a preference for pinniped hosts. Importantly, given the close proximity between seals, humans and livestock in many areas, there is the potential for cross-species infections.Peer reviewe

Not just fat : investigating novel physiological state biomarkers in cetacean blubber

Despite its importance as a predictor of fitness, no consensus has been reached about how to assess the nutritive body condition of free-ranging cetaceans. Standard indices of condition used in terrestrial ecology were assessed in cetaceans using data and samples from stranded animals. The most appropriate morphometric indices were identified as mass/length2 and girth/length (where mass data were unavailable). Blubber thickness, and blubber lipid content were poor indicators of condition. Variation in lipid content suggested that there may be tradeoffs occurring between different blubber functions involved in energy storage, controlling buoyancy, and preserving thermoregulatory properties in species with different life-history strategies. Novel blubber biomarkers of physiological state were investigated as objective and widely applicable tools for estimating body condition. Blubber cortisol concentrations were negatively correlated with morphometric indices of condition in stranded animals from two cetacean families. Blubber cortisol concentrations were thus identified as a candidate biomarker. When applied to a 13-year dataset of biopsy samples from female humpback whales (Megaptera novaeangliae), cortisol concentrations showed significant inter-annual variation. Concentrations were weakly correlated with annual pregnancy rates. High individual variability in blubber cortisol concentrations was likely a result of the multi-functional nature of cortisol. Cortisol concentrations thus probably provide a ‘snap shot’ of the metabolic state of the tissue when sampled. A proteomic approach was developed to extract and identify proteins in blubber. A range of proteins involved in numerous metabolic processes and pathways were identified. These proteins likely capture the range of physiological processes experienced by individuals at the time of sampling. This new proteomic approach will help to assign novel functions to marine mammal blubber in keeping with current understanding of the multi-functional role of adipose tissue as an endocrine organ in mammals. It will also allow the future development of protein biomarkers of health and physiological state

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

Overcoming the challenges of studying conservation physiology in large whales : a review of available methods

Large whales are subjected to a variety of conservation pressures that could be better monitored and managed if physiological information could be gathered readily from free-swimming whales. However, traditional approaches to studying physiology have been impractical for large whales, because there is no routine method for capture of the largest species and there is presently no practical method of obtaining blood samples from free-swimming whales. We review the currently available techniques for gathering physiological information on large whales using a variety of non-lethal and minimally invasive (or non-invasive) sample matrices. We focus on methods that should produce information relevant to conservation physiology, e.g. measures relevant to stress physiology, reproductive status, nutritional status, immune response, health, and disease. The following four types of samples are discussed: faecal samples, respiratory samples (‘blow’), skin/blubber samples, and photographs. Faecal samples have historically been used for diet analysis but increasingly are also used for hormonal analyses, as well as for assessment of exposure to toxins, pollutants, and parasites. Blow samples contain many hormones as well as respiratory microbes, a diverse array of metabolites, and a variety of immune-related substances. Biopsy dart samples are widely used for genetic, contaminant, and fatty-acid analyses and are now being used for endocrine studies along with proteomic and transcriptomic approaches. Photographic analyses have benefited from recently developed quantitative techniques allowing assessment of skin condition, ectoparasite load, and nutritional status, along with wounds and scars from ship strikes and fishing gear entanglement. Field application of these techniques has the potential to improve our understanding of the physiology of large whales greatly, better enabling assessment of the relative impacts of many anthropogenic and ecological pressures.Publisher PDFPeer reviewe

Sexual segregation and stable pregnancy rates in the Gulf of St. Lawrence’s minke whales Balaenoptera acutorostrata amidst environmental changes

Evaluating the health of baleen whale populations is crucial for understanding howenvironmental changes impact these top predators. Methodological advances, particularly in endocrine profiling, have enabled us to measure reproductive rates of populations as a proxy for population health. The Gulf of St. Lawrence (GSL), Canada, is an important summer feedingground for various North Atlantic baleen whale species and has undergone major ecosystem changes in recent decades. To explore the potential impacts on population health of minke whales Balaenoptera acutorostrata, we combined genetic analyses, endocrine profiling, and environmentaldata on prey availability to investigate population demographics and possible drivers of pregnancyrates between 2007 and 2015. Biopsy samples collected between May and October were sexed (n =187) using PCR, revealing a strong female bias (88.2%). Pregnancy status was determined throughblubber progesterone quantification, with progesterone concentrations of 0.061–8.04 ng g–1 fornon-pregnant individuals and 10.02–359.73 ng g–1 for pregnant individuals. High annual pregnancy rates were observed, ranging from 60 to 89% (mean: 74 ± 10%), with no consistent trenddetected over the study period. Generalised linear model results suggested species-specific prey availability in the year prior to pregnancy did not explain annual variation in pregnancy rates. We posit that this is due to the generalist feeding behaviour of minke whales. The results presented here indicate minke whales in the GSL exhibit sex-specific and reproductive spatial segregation.These pregnant females are likely using the area as a feeding ground prior to giving birth, with sufficient behavioural plasticity to withstand fluctuating food availability

Estimating the risks of exposure to harmful algal toxins among Scottish harbour seals

This research was supported by funding from the Scottish Government, Marine Scotland, Marine Mammal Scientific Support Research Programme MMSS/002/15 and by NERC National Capability NPG funding, grant No. NE/R015007/1.Harmful algal bloom (HAB) toxins consumed by marine predators through fish prey can be lethal but studies on the resulting population consequences are lacking. Over the past approximately 20 years there have been large regional declines in some harbour seal populations around Scotland. Analyses of excreta (faeces and urine from live and dead seals and faecal samples from seal haulout sites) suggest widespread exposure to toxins through the ingestion of contaminated prey. A risk assessment model, incorporating concentrations of the two major HAB toxins found in seal prey around Scotland (domoic acid (DA), and saxitoxins (STX)), the seasonal persistence of the toxins in the fish and the foraging patterns of harbour seals were used to estimate the proportion of adults and juveniles likely to have ingested doses above various estimated toxicity thresholds. The results were highly dependent on toxin type, persistence, and foraging regime as well as age class, all of which affected the proportion of exposed animals exceeding toxicity thresholds. In this preliminary model STX exposure was unlikely to result in mortalities. Modelled DA exposure resulted in doses above an estimated lethal threshold of 1900 µg/kg body mass affecting up to 3.8% of exposed juveniles and 5.3% of exposed adults. Given the uncertainty in the model parameters and the limitations of the data these conclusions should be treated with caution, but they indicate that DA remains a potential factor involved in the regional declines of harbour seals. Similar risks may be experienced by other top predators, including small cetaceans and seabirds that feed on similar prey in Scottish waters.Peer reviewe

Compound specific isotope analyses of harp seal teeth : tools for trophic ecology reconstruction

This work resulted from the ARISE project (NE/P006035/1 awarded to CM and RJ, and NE/P00623X/1 awarded to SS), part of the Changing Arctic Ocean programme, funded by the UKRI Natural Environment Research Council (NERC).As sentinels of ecosystem health, high trophic level predators integrate information through all levels of the food web. Their tissues can be used to investigate spatiotemporal variability in foraging behaviour, and with the appropriate analytical methods and tools, archived samples can be used to reconstruct past trophic interactions. Harp seal (Pagophilus groenlandicus) teeth collected in the 1990s from the Northwest Atlantic were analysed for bulk stable carbon and nitrogen isotopes (δ13Cbulk and δ15Nbulk), and compound specific stable nitrogen isotopes of amino acids (δ15NAA) for the first time. We developed a fine-scale, annual growth layer group (GLG) dentine sub-sampling method corresponding to their second and third year of life. In accordance with previous diet studies, while there was individual variability in δ15Nbulk, δ13Cbulk, and δ15NAA measurements, we did not detect significant differences in isotopic niche widths between males and females, or between GLGs. Relative trophic position was calculated as the baseline corrected δ15NAA values using trophic (glutamic acid) and source (phenylalanine and glycine) amino acids. Variability was measured between individuals in their relative trophic position, but within individual variability was low, suggesting that they fed at the same trophic level over these 2 years of life. These novel δ15NAA data may therefore suggest individual, specialist harp seal foraging behaviour in sub-adults. Our findings show that compound specific stable isotope signatures of archived, inert predator tissues can be used as tools for the retrospective reconstruction of trophic interactions on broad spatiotemporal scales.PostprintPeer reviewe

Toxic methaemoglobinaemia due to the action of nitrites

Opisane su methemoglobinemije prouzrokovane nitritima s naročitim obzirom na kemijske konzervanse u industriji mesnih proizvoda, koji su glavni izvor alimentarnih otrovanja nitritima. Iznesen je kazuistički prikaz nekoliko slučajeva otrovanja radnika u mesnoj industriji, koji su zabunom uzeli toksičke doze nitrita.Seven cases of toxic methaemoglobinaemia due to accidental nitrite poisoning in workers engaged in the preparation of meat and meat products are reported. In all these cases the meat-pickling salt was ingested as a consequence of the accidental water and food contamination. The main sources of nitrites in clinical intoxications are listed and the importance of the meat preserving solution for mass poisoning is emphasized. The general chemistry of the meat curing process is therefore briefly described. The clinical picture and the treatment of toxic methaemoglobinaemia are discussed