Metabolomics approach for predicting stomach and colon contents in dead Arctocephalus pusillus pusillus, Arctocephalus tropicalis, Lobodon carcinophaga and Ommatophoca rossii from sub-Antarctic region.

The dietary habits of seals play a pivotal role in shaping management and administration policies, especially in regions with potential interactions with fisheries. Previous studies have utilized various methods, including traditional approaches, to predict seal diets by retrieving indigestible prey parts, such as calcified structures, from intestines, feces, and stomach contents. Additionally, methods evaluating nitrogen and stable isotopes of carbon have been employed. The metabolomics approach, capable of quantifying small-scale molecules in biofluids, holds promise for specifying dietary exposures and estimating disease risk. This study aimed to assess the diet composition of five seal species-Arctocephalus pusillus pusillus, Lobodon carcinophaga, Ommatophoca rossii, and Arctocephalus tropicalis 1 and 2-by analyzing stomach and colon contents collected from stranded dead seals at various locations. Metabolite concentrations in the seal stomach and colon contents were determined using Nuclear Magnetic Resonance Spectroscopy. Among the colon and stomach contents, 29 known and 8 unknown metabolites were identified. Four metabolites (alanine, fumarate, lactate, and proline) from stomach contents and one metabolite (alanine) from colon contents showed no significant differences between seal species (p>0.05). This suggests that traces of these metabolites in the stomach and colon contents may be produced by the seals' gut microbiome or derived from other animals, possibly indicating reliance on fish caught at sea. Despite this insight, the cause of death for stranded seals remains unclear. The study highlights the need for specific and reliable biomarkers to precisely indicate dietary exposures across seal populations. Additionally, there is a call for the development of relevant metabolite and disease interaction networks to explore disease-related metabolites in seals. Ultimately, the metabolomic method employed in this study reveals potential metabolites in the stomach and colon contents of these seal species.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Circadian coupling of mitochondria in a deep-diving mammal.

Regulation of mitochondrial oxidative phosphorylation is essential to match energy supply to changing cellular energy demands, and to cope with periods of hypoxia. Recent work implicates the circadian molecular clock in control of mitochondrial function and hypoxia sensing. Because diving mammals experience intermittent episodes of severe hypoxia, with diel patterning in dive depth and duration, it is interesting to consider circadian-mitochondrial interaction in this group. Here, we demonstrate that the hooded seal (Cystophora cristata), a deep-diving Arctic pinniped, shows strong daily patterning of diving behaviour in the wild. Cultures of hooded seal skin fibroblasts exhibit robust circadian oscillation of the core clock genes per2 and arntl. In liver tissue collected from captive hooded seals, expression of arntl was some 4-fold higher in the middle of the night than in the middle of the day. To explore the clock-mitochondria relationship, we measured the mitochondrial oxygen consumption in synchronized hooded seal skin fibroblasts and found a circadian variation in mitochondrial activity, with higher coupling efficiency of complex I coinciding with the trough of arntl expression. These results open the way for further studies of circadian-hypoxia interactions in pinnipeds during diving.

Diverse papillomaviruses identified from Antarctic fur seals, leopard seals and Weddell seals from the Antarctic.

Papillomaviruses (family Papillomaviridae) are non-enveloped, circular, double-stranded DNA viruses known to infect squamous and mucosal epithelial cells. In the family Papillomaviridae there are 53 genera and 133 viral species whose members infect a variety of mammalian, avian, reptilian, and fish species. Within the Antarctic context, papillomaviruses (PVs) have been identified in Adélie penguins (Pygoscelis adeliae, 2 PVs), Weddell seals (Leptonychotes weddellii, 7 PVs), and emerald notothen (Trematomus bernacchii, 1 PV) in McMurdo Sound and Ross Island in eastern Antarctica. Here we identified 13 diverse PVs from buccal swabs of Antarctic fur seals (Arctocephalus gazella, 2 PVs) and leopard seal (Hydrurga leptonyx, 3 PVs) in western Antarctica (Antarctic Peninsula), and vaginal and nasal swabs of Weddell seals (8 PVs) in McMurdo Sound. These PV genomes group into four genera representing 11 new papillomavirus types, of which five are from two Antarctic fur seals and a leopard seal and six from Weddell seals.

Foraging habits of Northwest Atlantic hooded seals over the past 30 years: Future habitat suitability under global warming.

The Arctic is a global warming 'hot-spot' that is experiencing rapid increases in air and ocean temperatures and concomitant decreases in sea ice cover. These environmental changes are having major consequences on Arctic ecosystems. All Arctic endemic marine mammals are highly dependent on ice-associated ecosystems for at least part of their life cycle and thus are sensitive to the changes occurring in their habitats. Understanding the biological consequences of changes in these environments is essential for ecosystem management and conservation. However, our ability to study climate change impacts on Arctic marine mammals is generally limited by the lack of sufficiently long data time series. In this study, we took advantage of a unique dataset on hooded seal (Cystophora cristata) movements (and serum samples) that spans more than 30 years in the Northwest Atlantic to (i) investigate foraging (distribution and habitat use) and dietary (trophic level of prey and location) habits over the last three decades and (ii) predict future locations of suitable habitat given a projected global warming scenario. We found that, despite a change in isotopic signatures that might suggest prey changes over the 30-year period, hooded seals from the Northwest Atlantic appeared to target similar oceanographic characteristics throughout the study period. However, over decades, they have moved northward to find food. Somewhat surprisingly, foraging habits differed between seals breeding in the Gulf of St Lawrence vs those breeding at the "Front" (off Newfoundland). Seals from the Gulf favoured colder waters while Front seals favoured warmer waters. We predict that foraging habitats for hooded seals will continue to shift northwards and that Front seals are likely to have the greatest resilience. This study shows how hooded seals are responding to rapid environmental change and provides an indication of future trends for the species-information essential for effective ecosystem management and conservation.

Contrasting trophic transfer patterns of cadmium and mercury in the Arctic marine food web of east Hudson Bay, Canada.

We investigated trophic transfer of cadmium (Cd) through an Arctic marine food web in Hudson Bay and compared it with mercury (Hg), a metal known to strongly biomagnify. We evaluated blue mussel, sea urchin, common eider, sculpin, Arctic cod, and ringed seal for the influence of dietary and biological variables on variation in Cd and Hg concentrations. Age and size influenced metal concentrations among individuals within a vertebrate species. Consumer carbon and sulfur isotope values were correlated with their Cd and Hg concentrations, indicating habitat-specific feeding influenced metal bioaccumulation. Trophic transfer patterns for Cd depended on the vertebrate tissue, with food web biodilution observed for the muscle but not the liver. Liver Cd concentrations were higher in ringed seal and some common eider relative to prey. In contrast, we observed mercury biomagnification for both tissues. Tissue- and species-specific physiology can explain discrepancies of Cd trophic transfer in this Arctic marine food web.

Anthropogenic debris in three sympatric seal species of the Western Antarctic Peninsula.

Litter pollution is a growing concern, including for Antarctica and the species that inhabit this ecosystem. In this study, we investigated the microplastic contamination in three seal species that inhabit the Western Antarctic Peninsula: crabeater (Lobodon carcinophaga), leopard (Hydrurga leptonyx) and Weddell (Leptonychotes weddellii) seals. Given the worldwide ubiquity of this type of contaminant, including the Southern Ocean, we hypothesized that the three seal species would present anthropogenic debris in their feces. We examined 29 scat samples of crabeater (n = 5), leopard (n = 13) and Weddell (n = 11) seals. The chemical composition of the items found were identified using micro-Raman and micro-FTIR spectroscopies. All the samples of the three species presented anthropic particles (frequency of occurrence - %FO - 100 %). Fibers were the predominant debris, but fragments and filaments were also present. Particles smaller than 5 mm (micro debris) were predominant in all the samples. Leopard seals ingested significantly larger micro-debris in comparison with the other seal species. The dominant color was black followed by blue and white. Micro-Raman and micro-FTIR Spectroscopies revealed the presence of different anthropogenic pigments such as reactive blue 238, Indigo 3600 and copper phthalocyanine (blue and green). Carbon black was also detected in the samples, as well as plastic polymers such as polystyrene, polyester and polyethylene terephthalate (PET), polyamide, polypropylene and polyurethane These results confirm the presence of anthropogenic contamination in Antarctic seals and highlight the need for actions to mitigate the effects and reduce the contribution of debris in the Antarctic ecosystem.

Survival and adaptation of Streptococcus phocae in host environments.

Marine mammals are sentinel species representing the "health" of our oceans on which we are dependent. There are many threats to marine mammals including infectious diseases that increase with climate change and pollution of the marine environment. Streptococcus phocae has frequently been isolated from diseased or dead marine mammals. However, its pathogenicity and contribution to disease in marine mammals is still unknown. As bacteria including (potential) pathogens has to deal with different host environments during colonization or infection, we investigated the survival of S. phocae in fresh porcine and phocid blood, in seawater and in the presence of macrophages and (epithelial) cells from harbor seals and pigs. Furthermore, we tested adherence on and invasion of different (marine) mammalian cells by S. phocae. Our results showed that S. phocae can survive in seawater for at least 11 and 28 days at 16°C and 4°C, respectively. It is able to grow in blood of harbor and grey seals, but not in porcine blood. Furthermore, S. phocae is adherent and invasive to cells from seals and pigs, while the portion of invasive cells was higher in seal derived cells. Macrophages of harbor seals were more efficient in killing S. phocae than porcine macrophages. Our results indicate that S. phocae has strategies enabling it to adapt to the marine environment and seal hosts.

Monk seals (Monachus monachus) in the Mediterranean Sea: The threat of organochlorine contaminants and polycyclic aromatic hydrocarbons.

The Mediterranean monk seal (Monachus monachus) is an emblematic species of conservation concern. Anthropogenic pressures have led to severe population declines and significant fragmentation of the remaining populations. Because of their close relationship with coastal areas, Mediterranean monk seals may be potentially exposed to pollution from agricultural sources, as well as from oil tanker spills. Although monitoring of pollution has long been considered a priority for this species, data on monk seal contamination levels are scarce. In this study, 55 blubber samples of all genders and age classes collected during necropsies (1995-2013) from seals of the Eastern Mediterranean subpopulation were analyzed for organochlorine compounds (OCs), i.e., hexachlorobenzene (HCB), polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDTs), and polycyclic aromatic hydrocarbons (PAHs). Overall, PCBs > DDTs ≫ PAHs > HCB in all samples. Results showed a significant downward trend over the 19-year period for DDTs and HCB. No marked pattern was found for PAHs, even though relative abundance of cancerogenic PAH fraction rose in recent years. PCB levels in subadult specimens increased noticeably over time despite worldwide ban. Our findings did not suggest recent releases of DDT or new pesticides (e.g., Dicofol) in the environment, but may indicate an improper disposal of ancient storages of PCBs or a remobilization from reservoirs. OC levels exceeded thresholds that are commonly associated with immunosuppression and reproductive impairment in other pinnipeds. Hence, OCs could be responsible of potential toxicological effects in this subpopulation. This study represents the first report on PAH and one of the few reports on OC bioaccumulation in Mediterranean monk seals. Based on the levels found in the blubber of monk seals from the Eastern Mediterranean, and given the known harmful effects associated to the prolonged exposure to these substances and the reduced long-term expectations of species recovery, regular monitoring is advocated.

Gene expression and trace elements in Greenlandic ringed seals (Pusa hispida).

Marine top predators such as ringed seals biomagnify environmental contaminants; and with the increasing human activities in the Arctic, ringed seals are exposed to biologically significant concentrations of trace elements resulting in reproductive impairment, immunosuppression, and neurological damages. Little is known about the molecular effects of heavy metals on these vulnerable apex predators suffering from a rapidly changing Arctic with significant loss of sea-ice. In the present study, concentrations of cadmium (Cd), mercury (Hg) and selenium (Se) were measured in liver of sixteen Greenlandic ringed seals (nine adults and seven subadults) together with molecular biomarkers involved in bio-transformation, oxidative stress, endocrine disruption and immune activity in blood and blubber. The concentrations of trace elements increased in the following order: Hg > Se > Cd with levels of mercury and selenium being highest in adults. Aryl hydrocarbon receptor nuclear translocator (ARNT), peroxisome proliferator activated receptor alpha (PPARα, estrogen receptor alpha (ESR1), thyroid hormone receptor alpha (TRα) and interleukin - 2 (IL-2) mRNA transcript levels were highest in blubber, while heat shock protein 70 (HSP70) and interleukin - 10 (IL-10) were significantly higher in blood. There were no significant correlations between the concentrations of trace elements and mRNA transcript levels suggesting that stressors other than the trace elements investigated are responsible for the changes in gene expression levels. Since Hg seems to increase in Greenlandic ringed seals, there is a need to re-enforce health monitoring of this ringed seal population.

Relationship between red blood cell lifespan and endogenous carbon monoxide in the common bottlenose dolphin and beluga.

Certain deep-diving marine mammals [i.e., northern elephant seal (Mirounga angustirostris), Weddell seal (Leptonychotes weddellii)] have blood carbon monoxide (CO) levels that are comparable with those of chronic cigarette smokers. Most CO produced in humans is a byproduct of heme degradation, which is released when red blood cells (RBCs) are destroyed. Elevated CO can occur in humans when RBC lifespan decreases. The contribution of RBC turnover to CO concentrations in marine mammals is unknown. Here, we report the first RBC lifespans in two healthy marine mammal species with different diving capacities and heme stores, the shallow-diving bottlenose dolphin (Tursiops truncatus) and deep-diving beluga whale (Delphinapterus leucas), and we relate the lifespans to the levels of CO in blood and breath. The belugas, with high blood heme stores, had the longest mean RBC lifespan compared with humans and bottlenose dolphins. Both cetacean species were found to have three times higher blood CO content compared with humans. The estimated CO production rate from heme degradation indicates some marine mammals may have additional mechanisms for CO production, or delay CO removal from the body, potentially from long-duration breath-holds.NEW & NOTEWORTHY This is the first study to determine the red blood cell lifespan in a marine mammal species. High concentrations of carbon monoxide (CO) were found in the blood of bottlenose dolphins and in the blood and breath of belugas compared with healthy humans. Red blood cell turnover accounted for these high levels in bottlenose dolphins, but there may be alternative mechanisms of endogenous CO production that are contributing to the CO concentrations observed in belugas.

Metacercariae in Coprolites of Seals of the Genus Pachyphoca (Cystophorinae, Phocidae) from the Late Miocene of the North Caucasus.

Coprolites of the seal Pachyphoca volkodavi from the Upper Miocene deposits (Kherson) of the Fortepyanka locality (Republic of Adygea) were studied. Two out of five seal coprolites contained a structure resembling fish muscle tissue and included objects with morphology of trematode metacercariae. Obviously, the larvae of the parasite got into the digestive tract of seals from the fish they ate. Microscopic analysis of the coprolites did not reveal helminth eggs, which indicates possible absence of infection.

In silico simulations and molecular descriptors to predict in vitro transactivation potencies of Baikal seal estrogen receptors by environmental contaminants.

Baikal seals (Pusa sibirica) are vulnerable to high levels of organic pollutants. Here, we evaluated the transactivation potencies of bisphenols (BPs) and hydroxylated polychlorinated biphenyls (OH-PCBs) via the Baikal seal estrogen receptor α and ß (bsERα and bsERß) using in vitro and in silico approaches. In vitro reporter gene assays showed that most BPs and OH-PCBs exhibited estrogenic activity with bsER sub-type-specific potency. Among the BPs tested, bisphenol AF showed the lowest EC50 for both bsERs. 4'-OH-CB50 and 4'-OH-CB30 showed the lowest EC50 among OH-PCBs tested for bsERα and bsERß, respectively. 4-((4-Isopropoxyphenyl)-sulfonyl)phenol, 4'-OH-CB72, and 4'-OH-CB121 showed weak bsERα-specific transactivation. Only 4-OH-CB107 did not affect both bsERs. In silico docking simulations revealed the binding affinities of these chemicals to bsERs and partially explained the in vitro results. Using the in silico simulations and molecular descriptors as explanatory variables and the in vitro results as objective variables, the quantitative structure-activity relationship (QSAR) models constructed for classification and regression accurately separated bsER-active compounds from non-active compounds and predicted the in vitro bsERα- and bsERß-transactivation potencies, respectively. The QSAR models also suggested that chemical polarity, van der Waals surface area, bridging atom structure, position of the phenolic-OH group, and ligand interactions with key residues of the ligand binding pocket are critical variables to account for the bsER transactivation potency of the test compounds. We also succeeded in constructing computational models for predicting in vitro transactivation potencies of mouse ERs in the same manner, demonstrating the applicability of our approach independent of species-specific responses.

Predicting PFAS and Hydrophilic Trace Organic Contaminant Transport in Black Carbon-Amended Engineered Media Filters for Improved Stormwater Runoff Treatment.

Improved stormwater treatment is needed to prevent toxic and mobile contaminant transport into receiving waters and allow beneficial use of stormwater runoff. In particular, safe capture of stormwater runoff to augment drinking water supplies is contingent upon removing dissolved trace organic contaminants (TrOCs) not captured by conventional stormwater control measures. This study builds upon a prior laboratory-based column study investigating biochar and regenerated activated carbon (RAC) amendment for removing hydrophilic trace organic contaminants (HiTrOCs) and poly- and perfluoroalkyl substances (PFASs) from stormwater runoff. A robust contaminant transport model framework incorporating time-dependent flow and influent concentration is developed and validated to predict HiTrOC and PFAS transport in biochar- and RAC-amended stormwater filters. Specifically, parameters fit using a sorption-retarded intraparticle pore diffusion transport model were validated using data further along the depth of the column and compared to equilibrium batch isotherms. The transport model and fitted parameters were then used to estimate the lifetime of a hypothetical stormwater filter in Seal Beach, CA, to be 35 ± 6 years for biochar- and 51 ± 17 years for RAC-amended filters, under ideal conditions with no filter clogging. This work offers insights on the kinetics of HiTrOC and PFAS transport within biochar and RAC filters and on the impact of filter design on contaminant removal performance and longevity.

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Mercury Bioaccumulation and Cortisol Interact to Influence Endocrine and Immune Biomarkers in a Free-Ranging Marine Mammal.

Mercury bioaccumulation from deep-ocean prey and the extreme life history strategies of adult female northern elephant seals (Mirounga angustirostris) provide a unique system to assess the interactive effects of mercury and stress on animal health by quantifying blood biomarkers in relation to mercury (skeletal muscle and blood mercury) and cortisol concentrations. The thyroid hormone thyroxine (tT4) and the antibody immunoglobulin E (IgE) were associated with mercury and cortisol concentrations interactively, where the magnitude and direction of the association of each biomarker with mercury or cortisol changed depending on the concentration of the other factor. For example, when cortisol concentrations were lowest, tT4 was positively related to muscle mercury, whereas tT4 had a negative relationship with muscle mercury in seals that had the highest cortisol concentrations. Additionally, we observed that two thyroid hormones, triiodothyronine (tT3) and reverse triiodothyronine (rT3), were negatively (tT3) and positively (rT3) associated with mercury concentrations and cortisol in an additive manner. As an example, tT3 concentrations in late breeding seals at the median cortisol concentration decreased by 14% across the range of observed muscle mercury concentrations. We also observed that immunoglobulin M (IgM), the pro-inflammatory cytokine IL-6 (IL-6), and a reproductive hormone, estradiol, were negatively related to muscle mercury concentrations but were not related to cortisol. Specifically, estradiol concentrations in late molting seals decreased by 50% across the range of muscle mercury concentrations. These results indicate important physiological effects of mercury on free-ranging apex marine predators and interactions between mercury bioaccumulation and extrinsic stressors. Deleterious effects on animals' abilities to maintain homeostasis (thyroid hormones), fight off pathogens and disease (innate and adaptive immune system), and successfully reproduce (endocrine system) can have significant individual- and population-level consequences.

Is the inhalation influence on the level of mercury and PAHs in the lungs of the baltic grey seal (Halichoerus grypusgrypus)?

For many decades, mercury (Hg) has been recognized as one of the most dangerous environmental pollutants that negatively affects the ecosystem, including human health. Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic, toxic and potentially carcinogenic compounds. The process of respiration in addition to dietary intake is a significant source of these compounds to the human or marine mammalian body. Therefore, the aim of this study was to determine the sources of PAHs and labile forms of mercury in the lungs of dead seals found in the southern Baltic Sea. Of the PAHs: pyrene, fluoranthene and chrysene showed the highest concentrations. Considering the content of individual Hg fractions, the highest percentage was characterized by Hg labile 1b (related to organic matter). In a few specimens, deviations from the trend described above were observed: a higher proportion of Hg labile 1a (mainly halide-bound forms of mercury than the mean value which may indicate their origin from aerosols). Hg concentrations increased with seal age due to bioaccumulation and biomagnification of Hg from food; therefore, adsorption of atmospheric mercury on alveoli is probably of decreasing importance with seal age. Ratios obtained: FLA/PYR <1; B(a)A/CHR <1; FLA/(PYR + FLA) < 0.4 indicate a petrogenic source. In contrast, high correlations of B(a)A, FLA and PYR and CHR with Hg suggest a common source of PAHs and mercury - from food. Conversely, the presence of pyrogenic (combustion-derived) benzo(a)pyrene in the lungs of these mammals could indicate a respiratory route of entry. Mercury and PAHs in the lungs of the seals studied were mainly of trophic origin, but the results presented here make the hypothesis of an airborne influx of Hg and PAHs into the lungs from marine mammals plausible. This is of particular importance in juveniles (pups), who, at the initial stage of life, spend time on land and do not obtain food on their own.

Consequences of in vitro 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.

DEVELOPMENT AND VALIDATION OF A NOVEL DUPLEX PROBE-HYBRIDIZATION QUANTITATIVE PCR FOR LYMPHOMA-ASSOCIATED MIROUNGINE GAMMAHERPESVIRUS 3 IN NORTHERN ELEPHANT SEALS (MIROUNGA ANGUSTIROSTRIS).

Recently, a novel gammaherpesvirus, miroungine gammaherpesvirus 3 (MirGHV3), was described in two juvenile elephant seals (Mirounga angustirostris) with diffuse large B-cell lymphoma. We developed and validated a quantitative (q)PCR for rapid detection of MirGHV3 and investigated its potential association with lymphoma. We developed a duplex probe-hybridization qPCR with MirGHV3 DNA polymerase (pol) as the target gene. Each primer-probe combination was cross-validated against the others. Interference was not seen when they were run in the same well as a duplex assay. Twenty-three samples from seven northern elephant seals were tested using the duplex assay. Viral DNA was detected by the assay in 9 of 9 (100%) tissues affected by lymphoma and in 6 of 14 (43%) samples from tissues unaffected by lymphoma. There was a strong correlation between viral copies detected with each of the assays (P=0.0002). Viral load was significantly higher in tissues affected by lymphoma than in those unaffected (P<0.0001). Excluding the virus-negative samples, viral load was still significantly higher in tissues affected by lymphoma than in those unaffected (P=0.0004). This is consistent with a potential role of MirGHV3 in oncogenesis in northern elephant seals, although more studies are needed to determine this definitively. The qPCR developed has utility for further investigations of MirGHV3.

Transcriptomes of Clusterin- and S100B-transfected neuronal cells elucidate protective mechanisms against hypoxia and oxidative stress in the hooded seal (Cystophora cristata) brain.

BACKGROUND: The hooded seal (Cystophora cristata) exhibits impressive diving skills and can tolerate extended durations of asphyxia, hypoxia and oxidative stress, without suffering from irreversible neuronal damage. Thus, when exposed to hypoxia in vitro, neurons of fresh cortical and hippocampal tissue from hooded seals maintained their membrane potential 4-5 times longer than neurons of mice. We aimed to identify the molecular mechanisms underlying the intrinsic neuronal hypoxia tolerance. Previous comparative transcriptomics of the visual cortex have revealed that S100B and clusterin (apolipoprotein J), two stress proteins that are involved in neurological disorders characterized by hypoxic conditions, have a remarkably high expression in hooded seals compared to ferrets. When overexpressed in murine neuronal cells (HN33), S100B and clusterin had neuroprotective effects when cells were exposed to hypoxia. However, their specific roles in hypoxia have remained largely unknown. METHODS: In order to shed light on potential molecular pathways or interaction partners, we exposed HN33 cells transfected with either S100B, soluble clusterin (sCLU) or nuclear clusterin (nCLU) to normoxia, hypoxia and oxidative stress for 24 h. We then determined cell viability and compared the transcriptomes of transfected cells to control cells. Potential pathways and upstream regulators were identified via Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). RESULTS: HN33 cells transfected with sCLU and S100B demonstrated improved glycolytic capacity and reduced aerobic respiration at normoxic conditions. Additionally, sCLU appeared to enhance pathways for cellular homeostasis to counteract stress-induced aggregation of proteins. S100B-transfected cells sustained lowered energy-intensive synaptic signaling. In response to hypoxia, hypoxia-inducible factor (HIF) pathways were considerably elevated in nCLU- and sCLU-transfected cells. In a previous study, S100B and sCLU decreased the amount of reactive oxygen species and lipid peroxidation in HN33 cells in response to oxidative stress, but in the present study, these functional effects were not mirrored in gene expression changes. CONCLUSIONS: sCLU and S100B overexpression increased neuronal survival by decreasing aerobic metabolism and synaptic signaling in advance to hypoxia and oxidative stress conditions, possibly to reduce energy expenditure and the build-up of deleterious reactive oxygen species (ROS). Thus, a high expression of CLU isoforms and S100B is likely beneficial during hypoxic conditions.