Genetic and historic evidence for climate-driven population fragmentation in a top cetacean predator: the harbour porpoises in European water.

Recent climate change has triggered profound reorganization in northeast Atlantic ecosystems, with substantial impact on the distribution of marine assemblages from plankton to fishes. However, assessing the repercussions on apex marine predators remains a challenging issue, especially for pelagic species. In this study, we use Bayesian coalescent modelling of microsatellite variation to track the population demographic history of one of the smallest temperate cetaceans, the harbour porpoise (Phocoena phocoena) in European waters. Combining genetic inferences with palaeo-oceanographic and historical records provides strong evidence that populations of harbour porpoises have responded markedly to the recent climate-driven reorganization in the eastern North Atlantic food web. This response includes the isolation of porpoises in Iberian waters from those further north only approximately 300 years ago with a predominant northward migration, contemporaneous with the warming trend underway since the 'Little Ice Age' period and with the ongoing retreat of cold-water fishes from the Bay of Biscay. The extinction or exodus of harbour porpoises from the Mediterranean Sea (leaving an isolated relict population in the Black Sea) has lacked a coherent explanation. The present results suggest that the fragmentation of harbour distribution range in the Mediterranean Sea was triggered during the warm 'Mid-Holocene Optimum' period (approx. 5000 years ago), by the end of the post-glacial nutrient-rich 'Sapropel' conditions that prevailed before that time.

Rise of oceanographic barriers in continuous populations of a cetacean: the genetic structure of harbour porpoises in Old World waters.

BACKGROUND: Understanding the role of seascape in shaping genetic and demographic population structure is highly challenging for marine pelagic species such as cetaceans for which there is generally little evidence of what could effectively restrict their dispersal. In the present work, we applied a combination of recent individual-based landscape genetic approaches to investigate the population genetic structure of a highly mobile extensive range cetacean, the harbour porpoise in the eastern North Atlantic, with regards to oceanographic characteristics that could constrain its dispersal. RESULTS: Analyses of 10 microsatellite loci for 752 individuals revealed that most of the sampled range in the eastern North Atlantic behaves as a 'continuous' population that widely extends over thousands of kilometres with significant isolation by distance (IBD). However, strong barriers to gene flow were detected in the south-eastern part of the range. These barriers coincided with profound changes in environmental characteristics and isolated, on a relatively small scale, porpoises from Iberian waters and on a larger scale porpoises from the Black Sea. CONCLUSION: The presence of these barriers to gene flow that coincide with profound changes in oceanographic features, together with the spatial variation in IBD strength, provide for the first time strong evidence that physical processes have a major impact on the demographic and genetic structure of a cetacean. This genetic pattern further suggests habitat-related fragmentation of the porpoise range that is likely to intensify with predicted surface ocean warming.

Harbor porpoise thyroids: histologic investigations and potential interactions with environmental factors.

The thyroid plays an important role in development and is of primary importance in metabolism and heat loss for cetaceans, including the harbor porpoise (Phocoena phocoena). Several studies have demonstrated that environmental contaminants can alter various aspects of thyroid function in mammals and may contribute to various histologic changes. The present study completes the data set of a 2006 study by Das et al., by performing histological and immunohistologic investigations on thyroids of 36 harbor porpoises from Belgian and United Kingdom waters. The number and mean diameter of follicles (mum) and the relative proportion of follicular, connective, and vascular tissue (%) were quantified in the thyroid gland of each individual. Interfollicular fibrosis has been observed in these thyroid glands, and the collective findings support the hypothesis of an endocrine disruption of thyroid function through organochlorinated compounds. Our study aimed also to reveal potential relationships between thyroid morphometric data and metal levels (Cd, Fe, Zn, Cu, Se, and Hg) using multivariate statistical analysis. The multiple regressions revealed statistically significant relationships between trace elements (cadmium, selenium, and copper) and thyroid fibrosis. The largely negative relationships are interesting findings but do not support the hypothesis that these elements have an adverse effect on thyroid morphometry. Further research is needed to understand the nature of any relationship between organochlorine and trace element exposure and thyroid gland morphology and function in harbor porpoises.

Long-term feeding ecology and habitat use in harbour porpoises Phocoena phocoena from Scandinavian waters inferred from trace elements and stable isotopes.

BACKGROUND: We investigated the feeding ecology and habitat use of 32 harbour porpoises by-caught in 4 localities along the Scandinavian coast from the North Sea to the Barents Sea using time-integrative markers: stable isotopes (delta13C, delta15N) and trace elements (Zn, Cu, Fe, Se, total Hg and Cd), in relation to habitat characteristics (bathymetry) and geographic position (latitude). RESULTS: Among the trace elements analysed, only Cd, with an oceanic specific food origin, was found to be useful as an ecological tracer. All other trace elements studied were not useful, most likely because of physiological regulation and/or few specific sources in the food web. The delta13C, delta15N signatures and Cd levels were highly correlated with each other, as well as with local bathymetry and geographic position (latitude). Variation in the isotopic ratios indicated a shift in harbour porpoise's feeding habits from pelagic prey species in deep northern waters to more coastal and/or demersal prey in the relatively shallow North Sea and Skagerrak waters. This result is consistent with stomach content analyses found in the literature. This shift was associated with a northward Cd-enrichment which provides further support to the Cd 'anomaly' previously reported in polar waters and suggests that porpoises in deep northern waters include Cd-contaminated prey in their diet, such as oceanic cephalopods. CONCLUSION: As stable isotopes and Cd provide information in the medium and the long term respectively, the spatial variation found, shows that harbour porpoises experience different ecological regimes during the year along the Scandinavian coasts, adapting their feeding habits to local oceanographic conditions, without performing extensive migration.

Immunomodulatory effects of estradiol and cadmium in adult female rats.

A wide range of toxic effects has been associated with cadmium (Cd) exposure in mammals. However, the physiological factors that modulate these effects have received limited attention. We have previously demonstrated that neonatal exposure of rats to Cd during lactation results in sex-specific immunotoxic effects in both juvenile and adult rats. The objectives of this study were to determine the effects of 17beta-estradiol (E(2)) on the immunotoxicity of Cd in female rats. We compared the effects of 28 days of exposure to 0, 5, and 25 ppm cadmium chloride (CdCl(2)) through drinking water on ovariectomized Sprague-Dawley rats and on ovariectomized rats with E(2) implant which mimicked the physiological level of E(2) in female rat. Our results clarify the control of important immune functions by E(2) at physiological level and demonstrate significant interactions between Cd and E(2) effects on the cytotoxic activity of natural killer cells and phagocytosis of splenic cells as well as on the total number of thymocytes and of the four subpopulations of the thymocytes as defined by the expression of the cell-surface markers CD4 and CD8. Cd and E(2) share several mechanisms of action that may account for these interactions. The estrogenic potential of Cd could also account for some of the observed effects. These interactions have to be taken into consideration in evaluating the risk of Cd immunotoxicity and the possible interactions with hormonal treatments.

Zn, Cu, Cd and Hg binding to metallothioneins in harbour porpoises Phocoena phocoena from the southern North Sea.

BACKGROUND: Harbour porpoises Phocoena phocoena from the southern North Sea are known to display high levels of Zn and Hg in their tissues linked to their nutritional status (emaciation). The question arises regarding a potential role of metallothioneins (MTs) with regard to these high metal levels. In the present study, metallothionein detection and associated Zn, Cd, Cu and Hg concentrations were investigated in the liver and kidney of 14 harbour porpoises collected along the Belgian coast. RESULTS: Metallothioneins seemed to play a key role in essential metal homeostasis, as they were shown to bind 50% of the total hepatic Zn and 36% of the total hepatic Cu concentrations. Renal MTs also participated in Cd detoxification, as they were shown to bind 56% of the total renal Cd. Hg was mainly found in the insoluble fraction of both liver and kidney. Concomitant increases in total Zn concentration and Zn bound to MTs were observed in the liver, whereas Zn concentration bound to high molecular weight proteins remained constant. Cu, Zn and Cd were accumulated preferentially in the MT fraction and their content in this fraction increased with the amount in the hepatocytosol. CONCLUSION: MTs have a key role in Zn and Cu homeostasis in harbour porpoises. We demonstrated that increasing hepatic Zn concentration led to an increase in Zn linked to MTs, suggesting that these small proteins take over the Zn overload linked to the poor body condition of debilitated harbour porpoises.

Trace metal concentrations in Posidonia oceanica of North Corsica (northwestern Mediterranean Sea): use as a biological monitor?

BACKGROUND: Within semi-closed areas like the Mediterranean Sea, anthropic wastes tend to concentrate in the environment. Metals, in particular, are known to persist in the environment and can affect human health due to accumulation in the food chain. The seagrass Posidonia oceanica, widely found in Mediterranean coastal waters, has been chosen as a "sentinel" to quantify the distribution of such pollutants within the marine environment. Using a technique similar to dendrochronology in trees, it can act as an indicator of pollutant levels over a timeframe of several months to years. In the present study, we measured and compared the levels of eight trace metals (Cr, Ni, Cu, Zn, As, Se, Cd, and Pb) in sheaths dated by lepidochronology and in leaves of shoots sampled from P. oceanica meadows collected from six offshore sites in northern Corsica between 1988 and 2004; in the aim to determine 1) the spatial and 2) temporal variations of these metals in these areas and 3) to compared these two types of tissues. RESULTS: We found low trace metal concentrations with no increase over the last decade, confirming the potential use of Corsican seagrass beds as reference sites for the Mediterranean Sea. Temporal trends of trace metal concentrations in sheaths were not significant for Cr, Ni, Cu, As or Se, but Zn, Cd, and Pb levels decreased, probably due to the reduced anthropic use of these metals. Similar temporal trends between Cu levels in leaves (living tissue) and in sheaths (dead tissue) demonstrated that lepidochronology linked with Cu monitoring is effective for surveying the temporal variability of this metal. CONCLUSION: Leaves of P. oceanica can give an indication of the metal concentration in the environment over a short time period (months) with good accuracy. On the contrary, sheaths, which gave an indication of changes over long time periods (decades), seem to be less sensitive to variations in the metal concentration in the environment. Changes in human consumption of metals (e.g., the reduction of Pb in fuel) are clearly reflected in both organs. These results confirm that P. oceanica is a good bioindicator of metals and a good biomonitor species for assessing Cu in the environment.

Sex-specific effects of neonatal exposures to low levels of cadmium through maternal milk on development and immune functions of juvenile and adult rats.

Cadmium (Cd) is a major environmental contaminant. Although immunotoxic effects have been associated with Cd exposure, the inconsistency of experimental results underlines the need of an experimental approach more closely related to environmental conditions. We investigated the effects of exposing neonatal Sprague-Dawley rats to environmentally relevant doses of Cd through maternal milk. Dams received 10 parts per billion (ppb) or 5 parts per million (ppm) Cd chloride (CdCl2) in drinking water from parturition until the weaning of the pups. Half of the offspring was sampled at weaning time. The remaining juvenile rats received water without addition of Cd until adulthood. Cd accumulation in kidneys of juvenile rats fed from dams exposed to Cd indicated the transfer of the metal from mother to pups through maternal milk. This neonatal exposure resulted in decreased body, kidney and spleen weights of just weaned females but not of males. This effect was more pronounced in the less exposed females fed from dams exposed to 10 ppb Cd, which also displayed lower hepatic metallothionein-1 (MT-1) mRNA levels. The effect of Cd exposure on body and organ weights did not persist to adulthood. In contrast, we observed gender-specific effects of neonatal Cd exposure on the cytotoxic activity of splenic NK-cells of both juvenile and adult rats. Cd also strongly inhibited the proliferative response of Con A-stimulated thymocytes in both male and female adult rats 5 weeks after the cessation of Cd exposure. These immunotoxic effects were observed at doses much lower than those reported to produce similar effects when exposure occurred during adulthood. In conclusion, neonatal exposures to environmentally relevant levels of Cd through maternal milk represent a critical hazard liable to lead to both transitory and persistent immunotoxic effects.

Posidonia oceanica meadow: a low nutrient high chlorophyll (LNHC) system?

BACKGROUND: In spite of very low nutrient concentrations in its vicinity - both column and pore waters-, the Posidonia oceanica of the Revellata Bay displays high biomass and productivity. We measured the nutrient fluxes from the sediment into the water enclosed among the leaf shoots ("canopy water") to determine if it is possible source of nutrients for P. oceanica leaves. RESULTS: During the summer, the canopy water appears to act as a nutrient reservoir for the plant. During that period, the canopy water layer displays both a temperature 0.5 degrees C cooler than the upper water column, and a much higher nutrient content, as shown in this work using a very simple original technique permitting to sample water with a minimal disturbance of the water column's vertical structure. Despite low nutrient concentrations in pore water, mean net fluxes were measured from the sediment to the canopy water. These fluxes are sufficient to provide 20% of the mean daily nitrogen and phosphorus requirement of the P. oceanica shoots. CONCLUSION: An internal cycling of nutrients from P. oceanica senescent leaves was previously noted as an efficient strategy to help face low nutrient availability. The present study points out a second strategy which consists in holding back, in the canopy, the nutrients released at the water-sediment interface. This process occurs when long leaves, during poor nutrient periods in the water column, providing, to P. oceanica, the possibility to develop, high biomass, high chlorophyll quantities in low nutrient environment (a Low Nutrients High Chlorophyll system).

Trace metal and stable isotope measurements (delta13C and delta15N) in the harbour porpoise Phocoena phocoena relicta from the Black Sea.

Stable carbon and nitrogen isotopes (delta13C and delta15N) and trace metals (Cd, Zn, Cu, Fe, Se, and Hg) were analysed in the tissues of 46 harbour porpoises (Phocoena phocoena relicta) caught in fishing nets along the Ukrainian coasts between 1997 and 1998. Mean delta13C values differed significantly between male and female harbour porpoises suggesting a trophic segregation between sexes with a more coastal distribution for females at least during their gestation and nursing periods. Hepatic Hg was correlated to delta13C measurements, reflecting a different exposure linked to coastal vs offshore feeding habitats. A geographical comparison with existing data from other regions showed general low levels of Hg, Cd, Cu and Zn in the tissues of harbour porpoises from the Black Sea compared to other Atlantic and North Sea areas.

Nitrogen dynamics in Posidonia oceanica cuttings: implications for transplantation experiments.

A (15)N tracer study was performed during an experimental transplantation trial of natural Posidonia oceanica cuttings. The experiment was done in situ at 17 m depth in the Revellata Bay (Calvi, NW Corsica, France). Despite high survival rates of transplants (>90%) after one year, the weight and the N content of transplants are significantly lower than those of reference plants. In absence of roots, the transplants are not able to meet their N requirement, because leaf uptake is insufficient to replenish the N lost during the natural leaf decay. This could constitute a major cause of long-term failure for transplantation experiments or natural re-colonisation processes. The increase of the (15)N content in the roots shows that the plant re-allocates the nitrogen of one organ (i.e. leaves, rhizomes) to ensure the growth of another (i.e. roots).

Environmental factors affecting thyroid function of wild sea bass (Dicentrarchuslabrax) from European coasts.

Thyroid functional status of wild fish in relation with the contamination of their environment deserves further investigation. We here applied a multi-level approach of thyroid function assessment in 87 wild sea bass collected near several estuaries: namely the Scheldt, the Seine, the Loire, the Charente and the Gironde. Thyroxine (T(4)) and triiodothyronine (T(3)) concentrations in muscle were analyzed by radioimmunoassay. The activity of hepatic enzymes involved in extrathyroidal pathways of thyroid hormone metabolism, viz. deiodination, glucuronidation and sulfatation were analyzed. Last, follicle diameter and epithelial cell heights were measured. We observed changes that are predicted to lead to an increased conversion of T(4)-T(3) and lowered thyroid hormone excretion. The changes in the metabolic pathways of thyroid hormones can be interpreted as a pathway to maintain thyroid hormone homeostasis. From all compounds tested, the higher chlorinated PCBs seemed to be the most implicated in this perturbation.

Presence and regulation of metallothioneins in peripheral blood leukocytes of grey seals.

Heavy metal residues have been reported at high levels in marine mammals. Although the immunotoxicity of these contaminants has been demonstrated in laboratory models, evaluation of their potential immunotoxicity for human and wildlife species is complicated by variables that modulate the immune response and the immunotoxic effects of xenobiotics under field conditions. Metallothioneins (MT) modulate the bioavaibility of physiological cations and the toxicity of heavy metals. Moreover, these proteins have been demonstrated to modulate immune functions. In the present study, we demonstrated a rapid and sustained increase of transcripts of the two major isoforms of MT (MT-1 and MT-2) in grey seal peripheral blood leukocytes (PBL) exposed in vitro to Zn. This increase in mRNA corresponds to an increase of MT proteins in PBL. However, we demonstrated a high heterogeneity among the three major PBL subpopulations (granulocytes, lymphocytes and monocytes). Monocytes appear to be the most sensitive cells to Zn or Cd exposure. Intracellular MT levels in PBL subpopulations were dependent not only on the duration of exposure and the concentration, but also on the metal species. Cd was more potent than Zn as an inducer of MT in lymphocytes but not in monocytes. Moreover, grey seal peripheral blood lymphocytes are less sensitive to Cd exposure than human lymphocytes. This noninvasive approach helps to better assess the risk of heavy metal exposure by considering the potential role of MT as modulators of immune response.

Levels and enantiomeric signatures of methyl sulfonyl PCB and DDE metabolites in livers of harbor porpoises (Phocoena phocoena) from the Southern North Sea.

The concentration of 26 methyl sulfonyl metabolites of polychlorinated biphenyls (MeSO2-PCBs) and of p,p'-DDE (MeSO2-DDE) were determined in 19 liver samples from harbor porpoises (Phocoena phocoena) stranded between 1997 and 2000 on the Belgian and French North Sea Coasts. The total concentration of MeSO2-PCBs ranged from 39 to 4221 ng/g lipid weight (lw) and were generally higher in adults (age > 2 yr, range 969-4,221 ng/g lw) than in juveniles (age < 2 yr, range 39-1815 ng/g lw). The concentrations of MeSO2-DDE were generally also higher in adults (21-96 ng/g lw) than in juveniles (0.5-60 ng/g lw). Congeners 3- and 4-MeSO2-CB101 were the dominating metabolites in all samples. Due to their preferential retention in the liver, the MeSO2-PCB congeners could be divided into two groups. The first group was dominated by the 3-MeSO2-PCB congeners and consisted of MeSO2-CB31, -CB49, -CB52, -CB87, and -CB101, which all have a 2,5-chlorine substitution in the phenyl ring containing the methyl sulfonyl group. The second group was dominated by the 4-MeSO2-PCB congeners and consisted of MeSO2-CB64, -CB91, -CB110, and -CB132, which all have a 2,3,6-chlorine substitution. The ratios of sum of PCBs/sum of MeSO2-PCBs and p,p'-DDE/MeSO2-DDE differed greatly between individual subjects and ranged from 15 to 419 and from 17 to 1088, respectively. The ratio between the precursor PCB congeners and their corresponding metabolites ranged from 0.6 (CB49) to 175 (CB174). Enantiomeric fractions (EFs) for MeSO2-PCB atropisomers, which include 3-MeSO2-CB132, 3-MeSO2-CB149, 4-MeSO2-CB149, 3-MeSO2-CB174, and 4-MeSO2-CB174, were also measured in 8 out of the 19 subjects. High enantiomeric excess (EF > 0.73 or EF < 0.23) for the measured chiral MeSO2-PCB congeners was found in all samples. This result may suggest that one atropisomer may be preferentially formed in harbor porpoises or that the atropisomers are retained in a highly selective manner.

Perfluorinated chemicals infiltrate ocean waters: link between exposure levels and stable isotope ratios in marine mammals.

This is the first study to report on concentrations of perfluorinated organochemicals (FOCs) in marine mammals stranded along the southern North Sea coast in relation to stable nitrogen and carbon isotope ratios (delta15N and delta13C). The presence of FOCs in top predators such as marine mammals would indicate a potential biomagnification of these compounds and their widespread occurrence. Liver and kidney tissues of nine marine mammal species have been sampled. Among all the measured FOCs compounds, PFOS (perfluorooctane sulfonate) was predominant in terms of concentration. The highest PFOS concentrations were found in the liver of harbor seal compared to white-beaked dolphin, harbor porpoise, gray seal, sperm whale, white-sided dolphin, striped dolphin, fin whale, and hooded seal. PFOS concentrations differed significantly between sexes and age classes in harbor porpoises. Stable isotope measurements (delta13C and delta15N) were used in this study to describe the behavior of contaminants in food webs. We found a significant (p < 0.05) linear relationship between PFOS concentrations in livers of harbor porpoises and both muscle delta13C and delta15N measurements. Harbor and gray seals and white-beaked dolphin, which displayed the highest trophic position, contained the highest PFOS levels, while offshore feeders such as sperm whales, fin whales, striped dolphin, and white-sided dolphin showed lower PFOS concentrations than inshore species.