Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics.

Anadromous salmonids begin life adapted to the freshwater environments of their natal streams before a developmental transition, known as smoltification, transforms them into marine-adapted fish. In the wild, smoltification is a photoperiod-regulated process, involving radical remodeling of gill function to cope with the profound osmotic and immunological challenges of seawater (SW) migration. While prior work has highlighted the role of specialized "mitochondrion-rich" cells (MRCs) and accessory cells (ACs) in delivering this phenotype, recent RNA profiling experiments suggest that remodeling is far more extensive than previously appreciated. Here, we use single-nuclei RNAseq to characterize the extent of cytological changes in the gill of Atlantic salmon during smoltification and SW transfer. We identify 20 distinct cell clusters, including known, but also novel gill cell types. These data allow us to isolate cluster-specific, smoltification-associated changes in gene expression and to describe how the cellular make-up of the gill changes through smoltification. As expected, we noted an increase in the proportion of seawater mitochondrion-rich cells, however, we also identify previously unknown reduction of several immune-related cell types. Overall, our results provide fresh detail of the cellular complexity in the gill and suggest that smoltification triggers unexpected immune reprogramming.

Photoperiod-dependent developmental reprogramming of the transcriptional response to seawater entry in Atlantic salmon (Salmo salar).

The developmental transition of juvenile salmon from a freshwater resident morph (parr) to a seawater (SW) migratory morph (smolt), known as smoltification, entails a reorganization of gill function to cope with the altered water environment. Recently, we used RNAseq to characterize the breadth of transcriptional change which takes place in the gill in the FW phase of smoltification. This highlighted the importance of extended exposure to short, winter-like photoperiods (SP) followed by a subsequent increase in photoperiod for completion of transcriptional reprogramming in FW and efficient growth following transfer to SW. Here, we extend this analysis to examine the consequences of this photoperiodic history-dependent reprogramming for subsequent gill responses upon exposure to SW. We use RNAseq to analyze gill samples taken from fish raised on the photoperiod regimes we used previously and then challenged by SW exposure for 24 hours. While fish held on constant light (LL) throughout were able to hypo-osmoregulate during a 24 hours SW challenge, the associated gill transcriptional response was highly distinctive from that in fish which had experienced a 7-week period of exposure to SP followed by a return to LL (SPLL) and had consequently acquired the characteristics of fully developed smolts. Fish transferred from LL to SP, and then held on SP for the remainder of the study was unable to hypo-osmoregulate, and the associated gill transcriptional response to SW exposure featured many transcripts apparently regulated by the glucocorticoid stress axis and by the osmo-sensing transcription factor NFAT5. The importance of these pathways for the gill transcriptional response to SW exposure appears to diminish as a consequence of photoperiod mediated induction of the smolt phenotype, presumably reflecting preparatory developmental changes taking place during this process.

Diversified regulation of circadian clock gene expression following whole genome duplication.

Across taxa, circadian control of physiology and behavior arises from cell-autonomous oscillations in gene expression, governed by a networks of so-called 'clock genes', collectively forming transcription-translation feedback loops. In modern vertebrates, these networks contain multiple copies of clock gene family members, which arose through whole genome duplication (WGD) events during evolutionary history. It remains unclear to what extent multiple copies of clock gene family members are functionally redundant or have allowed for functional diversification. We addressed this problem through an analysis of clock gene expression in the Atlantic salmon, a representative of the salmonids, a group which has undergone at least 4 rounds of WGD since the base of the vertebrate lineage, giving an unusually large complement of clock genes. By comparing expression patterns across multiple tissues, and during development, we present evidence for gene- and tissue-specific divergence in expression patterns, consistent with functional diversification of clock gene duplicates. In contrast to mammals, we found no evidence for coupling between cortisol and circadian gene expression, but cortisol mediated non-circadian regulated expression of a subset of clock genes in the salmon gill was evident. This regulation is linked to changes in gill function necessary for the transition from fresh- to sea-water in anadromous fish. Overall, this analysis emphasises the potential for a richly diversified clock gene network to serve a mixture of circadian and non-circadian functions in vertebrate groups with complex genomes.

Arctic charr brain transcriptome strongly affected by summer seasonal growth but only subtly by feed deprivation.

BACKGROUND: The Arctic charr (Salvelinus alpinus) has a highly seasonal feeding cycle that comprises long periods of voluntary fasting and a short but intense feeding period during summer. Therefore, the charr represents an interesting species for studying appetite-regulating mechanisms in fish. RESULTS: In this study, we compared the brain transcriptomes of fed and feed deprived charr over a 4 weeks trial during their summer feeding season. Despite prominent differences in body condition between fed and feed deprived charr at the end of the trial, feed deprivation affected the brain transcriptome only slightly. In contrast, the transcriptome differed markedly over time in both fed and feed deprived charr, indicating strong shifts in basic cell metabolic processes possibly due to season, growth, temperature, or combinations thereof. The GO enrichment analysis revealed that many biological processes appeared to change in the same direction in both fed and feed deprived fish. In the feed deprived charr processes linked to oxygen transport and apoptosis were down- and up-regulated, respectively. Known genes encoding for appetite regulators did not respond to feed deprivation. Gene expression of Deiodinase 2 (DIO2), an enzyme implicated in the regulation of seasonal processes in mammals, was lower in response to season and feed deprivation. We further found a higher expression of VGF (non-acronymic) in the feed deprived than in the fed fish. This gene encodes for a neuropeptide associated with the control of energy metabolism in mammals, and has not been studied in relation to regulation of appetite and energy homeostasis in fish. CONCLUSIONS: In the Arctic charr, external and endogenous seasonal factors for example the increase in temperature and their circannual growth cycle, respectively, evoke much stronger responses in the brain than 4 weeks feed deprivation. The absence of a central hunger response in feed deprived charr give support for a strong resilience to the lack of food in this high Arctic species. DIO2 and VGF may play a role in the regulation of energy homeostasis and need to be further studied in seasonal fish.

Mandibular shape in farmed Arctic foxes (Vulpes lagopus) exposed to persistent organic pollutants.

We investigated if dietary exposure to persistent organic pollutants (POPs) affect mandibular asymmetry and periodontal disease in paired male-siblings of Arctic foxes (Vulpes lagopus). During ontogeny, one group of siblings was exposed to the complexed POP mixture in naturally contaminated minke whale (Balaenoptere acutorostarta) blubber (n = 10), while another group was given wet feed based on pig (Sus scrofa) fat as a control (n = 11). The ∑POP concentrations were 802 ng/g ww in the whale-based feed compared to 24 ng/g ww in the control diet. We conducted a two-dimensional geometric morphometric (GM) analysis of mandibular shape and asymmetry in the foxes and compared the two groups. The analyses showed that directional asymmetry was higher than fluctuating asymmetry in both groups and that mandibular shape differed significantly between the exposed and control group based on discriminant function analysis (T2 = 58.52, p = 0.04, 1000 permutations). We also found a non-significantly higher incidence of periodontal disease (two-way ANOVA: p = 0.43) and greater severity of sub-canine alveolar bone deterioration similar to periodontitis (two-way ANOVA: p = 0.3) in the POP-exposed group. Based on these results, it is possible that dietary exposure to a complexed POP mixture lead to changes in jaw morphology in Arctic foxes. This study suggests that extrinsic factors, such as dietary exposure to POPs, may affect mandibular shape and health in a way that could be harmful to wild Arctic populations. Therefore, further studies using GM analysis as an alternative to traditional morphometric methods should be conducted for wild Arctic fox populations exposed to environmental contaminants.

Photoperiod revisited: is there a critical day length for triggering a complete parr-smolt transformation in Atlantic salmon Salmo salar?

The present study investigated whether there is a critical length of photoperiod needed to stimulate a completed parr-smolt transformation (PST) in Atlantic salmon Salmo salar. In two experiments, S. salar parr of the Norwegian aquaculture strain held on continuous light were exposed to a short photoperiod (6 L:18D) followed by exposure to 8 L:16D, 12 L:12D, 16 L:8D, 20 L:4D and 24 L:0D in experiment 1 or to 6 L:18D followed by maintenance on 6 L:18D or exposure to 12 L:12D and 24 L:0D photoperiods in experiment 2. All groups, irrespective of photoperiod treatment, developed improved hypo-osmoregulatory ability. However, the development was greatest in the groups exposed to 20 L:4D and 24 L:0D in experiment 1 and 24 L:0D in experiment 2. In experiment 2, gill Na+ - K+ -ATPase activity increased in the group exposed to 24 L:0D, but not in the groups exposed to 12 L:12D and 6 L:18D. The groups exposed to 20 L:4D and 24 L:0D in experiment 1 and 24 L:0D in experiment 2 also grew better than fish exposed to shorter photoperiods. In experiment 2 only the group exposed to 24 L:0D showed a decrease in condition factor and increases in plasma growth hormone and brain type 2 deiodinase mRNA abundance. Hence, only the groups exposed to photoperiods above 16 L:8D developed classical smolt indices in the present experiment, leading us to conclude that the photoperiod increase needs to exceed 16 h daylight for stimulating a complete PST in the S. salar used in the present study.

Lifelong Exposure to PCBs in the Remote Norwegian Arctic Disrupts the Plasma Stress Metabolome in Arctic Charr.

Lake Ellasjøen on the remote Norwegian island of Bjørnøya is populated by Arctic charr (Salvelinus alpinus) having 20-fold higher body burdens of polychlorinated biphenyls (PCB) compared to charr from the neighboring Lake Laksvatn. This provides a natural setting to test the hypothesis that lifelong exposure to PCBs compromises the energy metabolism in this northernmost living salmonid. To test this, blood was sampled from charr from both lakes immediately after capture and following a 1 h handling and confinement stressor to assess possible differences in their energy metabolism and energy substrate mobilization, respectively. The plasma metabolome of charr was assessed by metabolite detection/separation with LC-MS. Plasma metabolite profiles revealed differences in key pathways involved in amino acid metabolism between charr from each lake, underscoring an impact of PCBs on energy metabolism in Arctic charr residing in Lake Ellasjøen. Subjecting charr from either lake to an acute stressor altered the plasma metabolite profiles and revealed distinct stress metabolome in Lake Ellasjøen charr, suggesting a reduced metabolic capacity. Taken together, lifelong exposure to PCBs in Ellasjøen charr disrupts the plasma metabolome, and may impair the adaptive metabolic response to stressors, leading to a reduced fitness.

Extended fasting does not affect the liver innate immune response in rainbow trout.

Activation of immune response pathway is energy demanding. We tested the hypothesis that negative energy balance will curtail the liver's capacity to evoke an immune response in rainbow trout (Oncorhynchus mykiss). Fish were either fed or fasted for 118 d and challenged with lipopolysaccharide (LPS) to determine the liver capacity to elicit an immune response. Fasting led to negative specific growth rate, reduced tissue metabolite levels, and higher transcript abundance of SOCS-2. LPS treatment increased the liver transcript abundances of IL-1ß and IL-8 and serum amyloid protein A, while SOCS-2 was reduced. LPS lowered plasma cortisol level only in the fasted fish, but did not affect liver glucocorticoid or mineralocorticoid receptor protein expressions. Extended fasting did not suppress the liver capacity to evoke an immune response. Upregulation of liver SOCS-2 may be playing a key role in the energy repartitioning, thereby facilitating immune response activation despite extended fasting in trout.

Biomarker response and hypothalamus-pituitary-interrenal axis functioning in Arctic charr from Bjørnøya (74°30' N), Norway, with high levels of organohalogenated compounds.

The populations of Arctic charr (Salvelinus alpinus) residing in Lake Ellasjøen at Bjørnøya Island in the Norwegian Arctic (74° 30'N, 19° 00'E) possess substantially higher levels of organohalogenated compounds (strongly dominated by polychlorinated biphenyls, PCBs) than conspecifics residing in other, proximate lakes on the island. In the present study we sampled large (<400g), immature charr from Lake Ellasjøen (high PCB levels) and Lake Laksvatn (reference lake, low PCB levels) by hook and line for an immediate blood sampling, and blood and tissue sampling after a 1h confinement stressor. This was done in order to investigate possible effects of pollutants on an acute stress performance in a high-latitude fish species by comparing muscle PCB levels, hepatic cytochrome P4501A (CYP1A) biomarker activation and functioning of the hypothalamus-pituitary-interrenal (HPI) axis between these two populations of Arctic charr. As expected sum PCB muscle levels were 8-fold higher on a wet weigh basis, and 19-fold higher on a lipid weight basis, in charr from Ellasjøen than in charr from Laksvatn. This was accompanied by a 3.5-fold higher liver cyp1a mRNA abundance in the Ellasjøen charr compared to Laksvatn charr. Brain transcript levels encoding glucocorticoid receptor 1 and 2 (GR2) and corticotropin-releasing factor, and pituitary transcript levels encoding GR2 and proopiomelanocortin A1 and A2 were higher in Ellasjøen charr than in Laksvatn charr, while interrenal transcript levels encoding melanocortin 2 receptor and steroidogenic acute regulatory protein were lower. There were no differences in plasma cortisol concentration between the two charr populations immediately after capture and one hour after confinement. The strong biomarker response to OHCs and altered mRNA abundances of key genes related to HPI axis functioning in the Ellasjøen charr suggest endocrine disruptive effects of OHCs in this charr population. Possible ecological implications are not known, but it cannot be excluded that a slower growth rate in Ellasjøen charr compared to Laksvatn charr due to an increased metabolic demand associated with the activation of xenobiotic defense and detoxification systems may have contributed to the lower body mass of Ellasjøen charr compared to Laksvatn charr.

Feedback from Arctic charr: Feed flavour stimulation and re-feeding after feed deprivation stimulate genes encoding both orexigenic and anorexigenic neuropeptides.

Despite vast research attention, the knowledge about central mechanisms of appetite regulation in teleost remains inconclusive. A common strategy in studies on appetite regulating mechanisms is to measure the response to feed restriction or - deprivation, but responses vary between fish species and between experiments, and are also likely dependent on the degree of energy perturbation. The anadromous Arctic charr is an interesting model for studying appetite regulation as its feeding cycle comprises months of winter anorexia, and hyperphagia during summer. Here we studied how the gene expression of putative hypothalamic appetite regulators were affected by two days, one week and one month feed deprivation during summer, and subsequent re-feeding and exposure to feed flavour. Short-term feed deprivation caused only a minor reduction in condition factor and had no effect on hypothalamic gene expression. Long-term feed-deprivation caused a marked reduction in weight and condition factor which contrasted the increase in weight and condition factor seen in ad libitum fed controls. A marked energy perturbation by feed deprivation was also indicated by a lower hypothalamic expression of the genes encoding insulin-like growth factor 1 (IGF1) and IGF1 binding protein 5 in the feed deprived charr compared to fed controls. Surprisingly, long-term feed deprivation and energy perturbation did not induce changes in hypothalamic appetite regulators. Unexpectedly, re-feeding and exposure to feed flavour caused an increase in the expression of the genes encoding the orexigenic agouti-related peptide and the anorexigenic melanocortin receptor 4 and cocaine- and amphetamine-regulated transcript. Our study gives strong evidence for a role of these in appetite regulation in Arctic charr, but their mechanisms of action remain unknown. We suggest that changes in gene expression are more likely to be registered during transition phases, e.g. from fasting to feeding and upon stimulatory inputs such as feed flavour.

Exposure to Persistent Organic Pollutants Reduces Testosterone Concentrations and Affects Sperm Viability and Morphology during the Mating Peak Period in a Controlled Experiment on Farmed Arctic Foxes (Vulpes lagopus).

We investigated testosterone production and semen parameters in farmed Arctic foxes by dietary exposure to persistent organic pollutants (POPs) for 22 months. Eight male foxes were given a diet of POP-contaminated minke whale blubber, whereas their eight male siblings were fed a control diet containing pig fat as the main fat source. The minke whale-based feed contained a ∑POPs concentration of 802 ng/g ww, whereas the pig-based feed contained ∑POPs of 24 ng/g ww. At the end of the experiment, ∑POP concentrations in adipose tissue were 8856 ± 2535 ng/g ww in the exposed foxes and 1264 ± 539 ng/g ww in the control foxes. The exposed group had 45-64% significantly lower testosterone concentrations during their peak mating season compared to the controls (p ≤ 0.05), while the number of dead and defect sperm cells was 27% (p = 0.07) and 15% (p = 0.33) higher in the exposed group. Similar effects during the mating season in wild Arctic foxes may affect mating behavior and reproductive success. On the basis of these results, we recommend testosterone as a sensitive biomarker of POP exposure and that seasonal patterns are investigated when interpreting putative endocrine disruption in Arctic wildlife with potential population-level effects.

NFAT5 genes are part of the osmotic regulatory system in Atlantic salmon (Salmo salar).

The anadromous Atlantic salmon utilizes both fresh and salt water (FW and SW) habitats during its life cycle. The parr-smolt transformation (PST) is an important developmental transition from a FW adapted juvenile parr to a SW adapted smolt. Physiological changes in osmoregulatory tissues, particularly the gill, are key in maintaining effective ion regulation during PST. Changes are initiated prior to SW exposure (preparative phase), and are completed when smolts enter the sea (activational phase) where osmotic stress may directly stimulate changes in gene expression. In this paper we identify 4 nuclear factor of activated T cells (NFAT5, an osmotic stress transcription factor) paralogues in Atlantic salmon, which showed strong homology in characterized functional domains with those identified in other vertebrates. Two of the identified paralogues (NFAT5b1 and NFAT5b2) showed increased expression following transfer from FW to SW. This effect was largest in parr that were maintained under short day photoperiod, and showed the highest increases in chloride ion levels in response to SW exposure. The results of this study suggest that NFAT5 is involved in the osmotic stress response of Atlantic salmon.

Effect of long-term fasting and a subsequent meal on mRNA abundances of hypothalamic appetite regulators, central and peripheral leptin expression and plasma leptin levels in rainbow trout.

Knowledge about neuroendocrine mechanisms regulating appetite in fish, including the role of leptin, is inconclusive. We investigated leptin mRNA abundance in various tissues, plasma leptin levels and the hypothalamic gene expression of putative orexigenic (neuropeptide Y and agouti-regulated peptide) and anorexigenic (melanocortin receptor, proopiomelanocortins (POMCs), cocaine- and amphetamine-regulated transcript and corticotropin-releasing factor) neuropeptides in relation to feeding status in rainbow trout (Oncorhynchus mykiss). Blood and tissues were first (Day 1) sampled from trout that had been fed or fasted for 4 months and the day after (Day 2) from fasted fish after they had been given a large meal, and their continuously fed counterparts. The fasted fish ate vigorously when they were presented a meal. There were no differences between fed, fasted and re-fed fish in hypothalamic neuropeptide transcript levels, except for pomca1 and pomcb, which were higher in fasted fish than in fed fish at Day 1, and which, for pomcb, decreased to the level in fed fish after the meal at Day 2. Plasma leptin levels did not differ between fasted, re-fed and fed fish. A higher leptina1 transcript level was seen in the belly flap of fasted fish than in fed fish, even after re-feeding on Day 2. The data do not reveal causative roles of the investigated brain neuropeptides, or leptin, in appetite regulation. It is suggested that the elevated pomc transcript levels provide a satiety signal that reduces energy expenditure during prolonged fasting. The increase in belly flap leptin transcript with fasting, which did not decrease upon re-feeding, indicates a tissue-specific role of leptin in long-term regulation of energy homeostasis.

Functional divergence of type 2 deiodinase paralogs in the Atlantic salmon.

Thyroid hormone (TH) is an ancestral signal linked to seasonal life history transitions throughout vertebrates. TH action depends upon tissue-localized regulation of levels of active TH (triiodothyronine, T3), through spatiotemporal expression of thyroid hormone deiodinase (dio) genes. We investigated the dio gene family in juvenile Atlantic salmon (Salmo salar) parr, which prepare for seaward migration in the spring (smoltification) through TH-dependent changes in physiology. We identified two type 2 deiodinase paralogs, dio2a and dio2b, responsible for conversion of thyroxine (T4) to T3. During smoltification, dio2b was induced in the brain and gills in zones of cell proliferation following increasing day length. Contrastingly, dio2a expression was induced in the gills by transfer to salt water (SW), with the magnitude of the response proportional to the plasma chloride level. This response reflected a selective enrichment for osmotic response elements (OREs) in the dio2a promoter region. Transcriptomic profiling of gill tissue from fish transferred to SW plus or minus the deiodinase inhibitor, iopanoic acid, revealed SW-induced increases in cellular respiration as the principal consequence of gill dio2 activity. Divergent evolution of dio2 paralogs supports organ-specific timing of the TH-dependent events governing the phenotypic plasticity required for migration to sea.

Tissue-specific molecular immune response to lipopolysaccharide challenge in emaciated anadromous Arctic charr.

Anadromous Arctic charr (Salvelinus alpinus) undergo voluntary winter fasting for months in the Arctic. We tested the hypothesis that extended fasting will compromise the ability of this species to evoke an immune response. Charr were either fed or fasted for 85 days and challenged with lipopolysaccharide (LPS), and the molecular immune response in the liver and spleen assessed at 8 and 96 h post-injection. LPS increased IL-1ß, IL-8, and serum amyloid protein A (SAA) mRNA levels in both groups, but the liver IL-1ß and IL-8, and spleen IL-8 responses were reduced in the fasted group. Fasting upregulated SOCS-1 and SOCS-2 mRNA abundance, while LPS stimulated SOCS-3 mRNA abundance and this response was higher in the fasted liver. Collectively, extended fasting and emaciation does not curtail the capacity of charr to evoke an immune response, whereas upregulation of SOCS may be a key adaptation to conserve energy by restricting the inflammatory response.

Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya.

Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate-Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.

Seasonal emaciation causes tissue redistribution and an increased potential for toxicity of lipophilic pollutants in farmed arctic fox (Vulpes lagopus).

Many Arctic animals carry high body burdens of organochlorine contaminants (OCs) as a result of long-range transport of persistent pollutants. It has been shown that seasonal mobilization of body fat in these species results in increased blood concentration of OCs. The authors investigated OC assimilation, tissue distribution, and biotransformation in farmed Arctic fox (Vulpes lagopus) continuously fed a diet containing contaminated minke whale blubber or lard (control) from 8 wk of age in August 2003, until sampling when they were at their fattest (in November 2004) and leanest (in June 2005). Markedly higher tissue (liver, adrenals, brain, and blood) OC levels were found in June than in November despite low exposure to OCs during emaciation, suggesting that OCs had been redistributed from adipose tissues to vital organs. There were no differences in the activities of hepatic biotransforming enzymes between exposed fat and control fat foxes, except for 16α-hydroxylation, which was higher in exposed fat foxes. In emaciated foxes, ethoxyresorufin activity was higher in exposed than in control foxes, indicating an enhanced potential for toxicity of OCs with emaciation. Lower activities of 6ß- and 2ß-hydroxylation were found in lean than in fat foxes, irrespective of OC treatment. The results show that emaciation increase the toxic potential of accumulated OCs and emphasize that body adiposity must be considered when time-trend analyses, risk assessments, and effect studies are designed. Environ Toxicol Chem 2013;32:1784-1792. © 2013 SETAC.

Dietary contaminant exposure affects plasma testosterone, but not thyroid hormones, vitamin A, and vitamin E, in male juvenile arctic foxes (Vulpes lagopus).

Levels of persistent organic pollutants (POP), such as polychlorinated biphenyls (PCB), are high in many Arctic top predators, including the Arctic fox (Vulpes lagopus). The aim of this study was to examine possible endocrine-disruptive effects of dietary POP exposure in male juvenile Arctic foxes in a controlled exposure experiment. The study was conducted using domesticated farmed blue foxes (Vulpes lagopus) as a model species. Two groups of newly weaned male foxes received a diet supplemented with either minke whale (Baleneoptera acutorostrata) blubber that was naturally contaminated with POP (exposed group, n = 5 or 21), or pork (Sus scrofa) fat (control group, n = 5 or 21). When the foxes were 6 mo old and had received the 2 diets for approximately 4 mo (147 d), effects of the dietary exposure to POP on plasma concentrations of testosterone (T), thyroid hormones (TH), thyroid-stimulating hormone (TSH), retinol (vitamin A), and tocopherol (viramin E) were examined. At sampling, the total body concentrations of 104 PCB congeners were 0.1 ± 0.03 µg/g lipid weight (l.w.; n = 5 [mean ± standard deviation]) and 1.5 ± 0.17 µg/g l.w. (n = 5) in the control and exposed groups, respectively. Plasma testosterone concentrations in the exposed male foxes were significantly lower than in the control males, being approximately 25% of that in the exposed foxes. There were no between-treatment differences for TH, TSH, retinol, or tocopherol. The results suggest that the high POP levels experienced by costal populations of Arctic foxes, such as in Svalbard and Iceland, may result in delayed masculine maturation during adolescence. Sex hormone disruption during puberty may thus have lifetime consequences on all aspects of reproductive function in adult male foxes.

Biotransformation of PCBs in Arctic seabirds: characterization of phase I and II pathways at transcriptional, translational and activity levels.

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO(2)-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher SigmaOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

Exposure and effects assessment of persistent organohalogen contaminants in arctic wildlife and fish.

Persistent organic pollutants (POPs) encompass an array of anthropogenic organic and elemental substances and their degradation and metabolic byproducts that have been found in the tissues of exposed animals, especially POPs categorized as organohalogen contaminants (OHCs). OHCs have been of concern in the circumpolar arctic for decades. For example, as a consequence of bioaccumulation and in some cases biomagnification of legacy (e.g., chlorinated PCBs, DDTs and CHLs) and emerging (e.g., brominated flame retardants (BFRs) and in particular polybrominated diphenyl ethers (PBDEs) and perfluorinated compounds (PFCs) including perfluorooctane sulfonate (PFOS) and perfluorooctanic acid (PFOA) found in Arctic biota and humans. Of high concern are the potential biological effects of these contaminants in exposed Arctic wildlife and fish. As concluded in the last review in 2004 for the Arctic Monitoring and Assessment Program (AMAP) on the effects of POPs in Arctic wildlife, prior to 1997, biological effects data were minimal and insufficient at any level of biological organization. The present review summarizes recent studies on biological effects in relation to OHC exposure, and attempts to assess known tissue/body compartment concentration data in the context of possible threshold levels of effects to evaluate the risks. This review concentrates mainly on post-2002, new OHC effects data in Arctic wildlife and fish, and is largely based on recently available effects data for populations of several top trophic level species, including seabirds (e.g., glaucous gull (Larus hyperboreus)), polar bears (Ursus maritimus), polar (Arctic) fox (Vulpes lagopus), and Arctic charr (Salvelinus alpinus), as well as semi-captive studies on sled dogs (Canis familiaris). Regardless, there remains a dearth of data on true contaminant exposure, cause-effect relationships with respect to these contaminant exposures in Arctic wildlife and fish. Indications of exposure effects are largely based on correlations between biomarker endpoints (e.g., biochemical processes related to the immune and endocrine system, pathological changes in tissues and reproduction and development) and tissue residue levels of OHCs (e.g., PCBs, DDTs, CHLs, PBDEs and in a few cases perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonates (PFSAs)). Some exceptions include semi-field studies on comparative contaminant effects of control and exposed cohorts of captive Greenland sled dogs, and performance studies mimicking environmentally relevant PCB concentrations in Arctic charr. Recent tissue concentrations in several arctic marine mammal species and populations exceed a general threshold level of concern of 1 part-per-million (ppm), but a clear evidence of a POP/OHC-related stress in these populations remains to be confirmed. There remains minimal evidence that OHCs are having widespread effects on the health of Arctic organisms, with the possible exception of East Greenland and Svalbard polar bears and Svalbard glaucous gulls. However, the true (if any real) effects of POPs in Arctic wildlife have to be put into the context of other environmental, ecological and physiological stressors (both anthropogenic and natural) that render an overall complex picture. For instance, seasonal changes in food intake and corresponding cycles of fattening and emaciation seen in Arctic animals can modify contaminant tissue distribution and toxicokinetics (contaminant deposition, metabolism and depuration). Also, other factors, including impact of climate change (seasonal ice and temperature changes, and connection to food web changes, nutrition, etc. in exposed biota), disease, species invasion and the connection to disease resistance will impact toxicant exposure. Overall, further research and better understanding of POP/OHC impact on animal performance in Arctic biota are recommended. Regardless, it could be argued that Arctic wildlife and fish at the highest potential risk of POP/OHC exposure and mediated effects are East Greenland, Svalbard and (West and South) Hudson Bay polar bears, Alaskan and Northern Norway killer whales, several species of gulls and other seabirds from the Svalbard area, Northern Norway, East Greenland, the Kara Sea and/or the Canadian central high Arctic, East Greenland ringed seal and a few populations of Arctic charr and Greenland shark.