Seasonal variation of histopathological and histochemical markers of PAH exposure in blue mussel (Mytilus edulis L.)

The aim of this work was to study seasonal variation of histopathological and histochemical markers in blue mussels (Mytilus edulis L.) exposed to pyrogenic PAH contaminants. Mussels were collected in January, June, September and October from a sampling site in the vicinity of the discharge from an aluminium smelter and from a clean reference site. Histopathological analysis was carried out on the digestive gland (DG) and the gonads, lipofuscin and neutral lipids were analysed in the DG. Clear responses in lipofuscin and neutral lipids were detected in the DG of mussels collected from the polluted site at some sampling times. Moreover, these mussels presented atrophy in digestive tubules and haemocytic aggregates in the gonad and DG. However, in all parameters studied, the magnitude of the response showed clear seasonal variation.     

Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis)

Acetylcholinesterase in mussel is potentially a useful biomarker of exposure to organophosphates (OP) in the marine environment. This study looked at cholinesterase activity in subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measurement of enzyme rates demonstrated that although highest specific activity was found in foot 'mitochondrial' fraction, recovery of activity was very low. Gill 'microsomal' fraction had the second highest specific activity with a useful level of recovery and therefore was the most suitable tissue fraction for biomarker applications. Comparative studies of alternative alkylthiocholine substrates and competitive inhibitors suggest there is a single cholinesterase enzyme type present in this fraction. Inhibition of alkylcholine hydrolysis by BW284C51, specific to acetylcholinesterase in vertebrates, showed that cholinesterase activity in gill 'microsomal' fraction is inhibited by this compound but to a lesser extent than in vertebrate AChE. Inhibition of cholinesterase activity by azamethiphos in gill 'microsomal' fraction gave an IC50 of approximately 100 microM and showed both time and concentration dependence. However this indicates a lower potency compared to other animals and it is debatable whether mussel cholinesterase activity is useful as a biomarker of exposure in the field.     

Effects of various pollutant mixtures on immune responses of the blue mussel (Mytilus edulis) collected at a salinity gradient in Danish coastal waters

The Baltic Sea is a semi-enclosed sea with a steady salinity gradient (3‰–30‰). Organisms have adapted to such low salinities, but are suspected to be more susceptible to stress. Within the frame of the integrated environmental monitoring BONUS + project “BEAST” the applicability of immune responses of the blue mussel was investigated in Danish coastal waters. The sampling sites were characterised by a salinity range (11–19‰) and different mixtures of contaminants (metals, PAHs and POPs), according to chemical analysis of mussel tissues. Variation partitioning (redundancy analysis) was applied to decompose salinity and contamination effects. The results indicated that cellular immune responses (total and differential haemocyte count, phagocytic activity and apoptosis) were mainly influenced by contaminants, whereas humoral factors (haemolytic activity) were mainly impacted by salinity. Hence, cellular immune functions may be suitable as biomarkers in monitoring programmes for the Baltic Sea and other geographic regions with salinity variances of the studied range.     

Variability of heat shock proteins and glutathione S-transferase in gill and digestive gland of blue mussel,Mytilus edulis

Glutathione S-transferase (GST) and heat shock proteins (hsps) 40, 60, 70 and 90 were determined by immunoblotting using actin as an internal control in Mytilus edulis from one station outside (site1) and three stations within (sites 2-4) Cork Harbour, Ireland. Comparisons were made between gill and digestive gland and between sites. Gill shows generally higher hsp 60, 70 and 90 while digestive gland has higher hsp 40. Site 1 showed higher gill hsps 40 and 70 than sites 2-4 while gill GST was higher in sites 3 and 4 than 1 and 2. Comparison with sites in the North Sea (site 5: outside Tj?rn? in The Koster archipelago in the Skagerack) and Baltic Sea (site 6: Ask? island) also revealed lower hsps 40 and 70 in site 6 (low salinity) than site 5 (high salinity) although hsps 60, 70 and 90 were detectable in digestive gland unlike sites 1-4. Previously, only hsp 70 had been studied at these sites [Mar. Environ. Res. 39. (1995), 181]. At the mRNA level, gill hsp 70 is 80-fold higher at Tj?rn? than Ask?. These data suggest that, while salinity may slightly decrease hsp 40 and 70, both hsp 70 and GST are selectively up-regulated by approx. 10- and 3-fold, respectively, at Tj?rn? compared to the other sites which we attribute to exposure to more widely fluctuating pollution levels.     

Nutrient regeneration by mussel Mytilus edulis spat assemblages in a macrotidal system

Besides exercising grazing control over phytoplankton populations, suspension-feeding bivalves can also stimulate carrying capacity by regeneration of nutrients. This study provides new data on nutrient uptake and release dynamics, and potential implications for availability and stoichiometry of nutrients, for Mytilus edulis spat collectors in the Netherlands. Uptake and release rates were measured in situ on intact spat collector ropes in a eutrophic macrotidal system in relation to development of ropes in terms of mussel biomass and associated components (fauna, flora, and organic material).There was a good fit between uptake/release rates and mussel weight based on allometric scaling functions, despite the occurrence of a substantial biomass of associated fauna, flora and organic matter on ropes. On a unit biomass basis, nutrient release rates were much higher than reported in other studies, which we attribute to greater activity of small mussels. Accounting for greater weight-specific activity of small mussels, spat collectors released more P than reported for other systems. We show that spat collectors can affect relative availabilities of N, P and Si, and we show that SMCs (Seed Mussel Collectors) likely stimulated phytoplankton production through regeneration of N and of Si, which were at limiting concentrations at different points in time. In the case of Si, stimulation would be restricted to diatoms. We conclude that SMCs are able to stimulate phytoplankton production rates, and thereby carrying capacity, and are able to influence phytoplankton composition.     

Multidrug resistance in the embryos and larvae of the mussel Mytilus edulis.

Cells exhibiting the multidrug resistance (MDR) phenotype demonstrate a decreased intracellular drug accumulation due to an active outward transport and decreased intracellular flux. This study demonstrates the inhibition of MDR in mussel (Mytilus edulis) embryos and larvae based on a simple bioassay. The development of embryos was assessed and abnormalities identified at key stages of development, including gastrulation, trochophore and prodissoconch stages. The incidence of developmental abnormalities was significantly increased in the presence of vinblastine, MMS, chloroquine, mitomycin-C, cadmium chloride and colchicine, compared to clean seawater. Consistently, there was a further increase in the number and severity of deformities observed when each toxin was added in the presence of verapamil. Larval growth was also significantly impaired in the presence of verapamil. Increased accumulation of fluorescent MDR dyes, such as rhodamine B, has been measured and shown to be verapamil sensitive. This bioassay encompasses a period of intense cellular activity during which the impairment of a number of critical processes results in abnormal growth and development.     

Numerical modelling of spatio-temporal variability of growth of Mytilus edulis (L.) and influence of its cultivation on ecosystem functioning

One of the key needs of the aquaculture industry is the implementation of effective management methods to ensure the sustainability, economic viability and minimization of negative impacts on both human and ecosystem well-being. The authors developed a Fortran 90 implementation of the dynamic energy budget (DEB) model for Mytilus edulis. The model has been further developed to include physiological interactions with the ecosystem and coupled to a biogeochemical nutrient–phytoplankton–zooplankton–detritus (NPZD) model. Phytoplankton and detritus uptakes, oxygen utilisation, CO₂ production, NH₄ excretion, egestion of faeces, and assimilation of food are modelled. A novel approach was derived that accounts for the allocation of C and N in mussel flesh and shell organic fraction. The DEB–NPZD model has been subsequently coupled to a high resolution three dimensional numerical coastal ocean model of the south–west coast of Ireland, where approximately 80% of national rope mussel is produced annually. Simulations have been carried out for the time period July 2010–June 2011, for which the field data on mussel biometrics and ambient seawater properties were collated. The model accurately reproduced the spatio-temporal variability in blue mussel growth. It is also shown that the ecosystem dynamics is affected by the presence of aquaculture farms. The modelling system presented allows for the assessment of the impacts of aquaculture activities on water quality, quantification of the production and ecological carrying capacities and improvement of our understanding of the ecosystem functioning with particular emphasis on interactions between various trophic levels.     

The effect of pollution on scope for growth of the mussel (Mytilus edulis L.) in Iceland

In order to evaluate effects of contaminants at high latitudes, the effects on scope for growth (SFG) were evaluated for both transplanted and resident blue mussel (Mytilus edulis L.) near large and small harbours in Iceland. Low SFG values were found for mussels at the mouth and in the inner part of the large Reykjavík harbour, which could be attributed to elevated levels of organotins and polyaromatic hydrocarbons. Mussels in smaller and/or less polluted harbours and sites off Reykjavík harbour only showed a slight reduction in SFG. No major effects were found at sites >3 km from the large harbour of Reykjavík. SFG provides valuable information on the effects of harbour pollutants in Iceland, in particular in areas of extensive shipping activity. The use of this biomarker on high latitude organisms is highly relevant due to extended oil exploration at higher latitudes.     

Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures.

Expanding industrial activities in the Arctic require an urgent assessment of the toxicity of chemicals at low temperatures. Organisms acclimatized to low temperature exhibit specific adaptations. For example, the amount of unsaturated lipids is increased to maintain the fluidity of the cell membranes. It has been hypothesized that such temperature-induced alterations in membrane lipid composition may affect the stability of lysosomal and cell membranes in the common mussel, Mytilus edulis, an organism exposed to seasonal temperature extremes. As mussels may be exposed to petroleum compounds along industrialized coastlines, we tested the combined effects of exposure to low temperature and the petroleum compound, phenanthrene, on haemocyte membrane stability. Test animals, acclimated to either 0 or 10 degrees C, were exposed to phenanthrene (0 = control or 500 micrograms l-1) and haemocytes were examined using the neutral red retention assay (lysosomal stability) and a fluorescence assay (cell membrane stability). At 0 degree C, lysosomal and cell membranes from uncontaminated mussels were destabilized compared with 10 degrees C (P = 0.0005). No significant effects (P > 0.05) of phenanthrene were detected at either temperature. Possible mechanisms underlying membrane destabilization include a weaker physical resistance of the membrane due to a higher amount of unsaturated lipids, a potentially higher level of reactive oxygen radicals at low temperature and the higher susceptibility of unsaturated lipids to oxidative stress. More work is required to better understand the consequences of this membrane destabilization at low temperature on the susceptibility of the organism to pollutants.     

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.     

Numbers of larvae and primary plantigrades of the mussel Mytilus edulis in the western Dutch Wadden Sea

Abundance of larvae and primary plantigrades in the blue mussel Mytilus edulis L. was studied quantitatively in the western part of the Dutch Wadden Sea. Observations were made in seven years between 1981 and 1996. In general, a first large peak of larvae appeared in May, followed by one or more much smaller peaks until September. For pelagic larvae an approximate life time of four to six weeks was calculated (on average about five weeks). Pelagic larvae appeared to have a relatively higher survival chance at low larval concentrations than at high concentrations. No relation could be demonstrated between time of larval peaks and spring tides. Year-to-year differences in abundance of plantigrades did not predict mussel recruitment success on nearby tidal flats.     

Aggregational behavior of the blue mussels Mytilus edulis and Mytilus trossulus: a potential pre‐zygotic reproductive isolation mechanism

The blue mussels Mytilus edulis and Mytilus trossulus occur sympatrically and are able to hybridize in populations on the eastern coast of Newfoundland, Canada, presenting an opportunity to study their aggregational behavior. Aggregation behavior may therefore provide insight into post‐settlement interactions and pre‐zygotic reproductive isolation between the species. Three treatments were designed using M. edulis and M. trossulus to investigate their intraspecific and interspecific spatial distribution patterns. With Ripley’s K‐function and Monte Carlo simulation analysis, we found that in the single‐species treatment, M. edulis aggregated significantly but not M. trossulus. Based on results of two‐way ANOVAs, both the number of aggregations and the moving distance were significantly affected by the treatments (single‐species or mixed‐species treatment) and times (24, 48, 72 and 96 h). In further pairwise comparisons using Tukey’s test, M. edulis aggregated differently with or without M. trossulus occupying the same tank, suggesting that the aggregational behavior of M. edulis could be driven by species‐specific chemical cues. The result that M. edulis aggregates intraspecifically may increase the probability of intraspecific fertilization of the spawned gametes and thus function as a pre‐zygotic reproductive isolation mechanism maintaining the blue mussel hybrid zone.     

Biochemical and histological responses in mussel (Mytilus edulis) exposed to North Sea oil and to a mixture of North Sea oil and alkylphenols

Several environmental chemicals are suspected to be responsible for adverse health effects on the reproductive system in various organisms. During this work, environmentally relevant concentrations of North Sea oil were used alone or in combination with alkylphenols and additional PAH to study the effect on vitellogenin-like protein expression and gonadal development in mussels. North Sea oil (0.5 ppm) induced the expression of phospho-proteins in both sexes indicating that some compounds are oestrogen-mimics. This induction was not seen in samples dosed with the mixture but signs of toxic effects were observed in the gonads. Indeed, numerous degenerating ovarian follicles in females and foci, similar to vertebrate melanomacrophage centres, were observed in testes.     

基于线粒体COI序列分析对紫贻贝群体遗传多样性的研究分析

本文采用线粒体COI基因序列分析对我国沿海6个紫贻贝群体124个个体的遗传多样性和群体结构进行了初步研究.在紫贻贝6个群体中共发现13个单倍型和33个核苷酸多态位点.青岛群体单倍型数目是最多的,温州群体、丹东群体和宁德群体次之.6个紫贻贝群体的基因多样性为0.569～0.821,核苷酸多样性为0.015 5～0.051...     

Immuno-localisations (GSSP) of subcellular accumulation sites of phenanthrene, aroclor 1254 and lead (Pb) in relation to cytopathologies in the gills and digestive gland of the mussel Mytilus edulis

Cell and tissue pathology of both, gill and digestive tissue, has been the subject of many studies for the elucidation of contaminant-induced biological effects. In the present study, cellular pathological alterations were linked to subcellular sites of chemical accumulation in gills and digestive gland tissues. For this purpose, mussels (Mytilus edulis) were exposed to the organic contaminants aroclor 1254 (PCB) (20 microg/L), phenanthrene (PAH) (150 microg/L) or the metal lead (Pb) (2.5mg/L). The localization of chemicals at the subcellular level was analysed by an antibody-based detection system (GSSP) by the use of commercially available antibodies specifically directed against the chemicals. Pathological changes were analysed in parallel in identical samples by transmission electron microscopy. After exposure to the different contaminants, cell organelles such as mitochondria, the endo-lysosomal system as well as endoplasmic reticulum showed clear evidence of chemically-induced alterations. Large numbers of crystalloid inclusions were found in mitochondria and in autophagic lysosomes as well as multi-lamellated whorls after PAH and aroclor exposure. Immunocytochemical detection of the chemicals showed their accumulation inside of various cell organelles such as lysosomes, mitochondria, and nuclei. Additionally, chemicals were localized in association to membranes, cilia and microvilli of gill and digestive gland cells. Furthermore, the chitinous rod and mucus secretions of gill epithelial cells were positively labelled for contaminants indicating their role in protection. Localization of contaminants by immuno-detection in combination with pathological diagnosis gives insights into the cellular targets of chemical attack.     

Tissue sampling from live blue mussels,Mytilus edulis. A field study from the Swedish west coast

Histological techniques are often used to study environmental effects on mussels, but since these techniques include killing of the individuals, rare or endangered populations cannot be studied using conventional tissue sampling. This study is an attempt to find a method that can be used repeatedly with the same mussel individual and which does not affect growth and survival. From 200 mussels, Mytilus edulis, tissue was sampled in different ways, such as drilling a hole in the shell or prising apart the shell valves. Two kind of instruments were used, an injection needle and surgery forceps. Some of the drilled mussels had their holes sealed again with cement.Drilling a hole in the shell, removing tissue sample with surgical forceps and then leaving the holes open did not seriously harm the mussels during the two months the experiment lasted. But if the holes were sealed with cement, both length and weight growth were negatively affected (35% lower length growth and 36% lower weight growth compared to the control mussels). Mortality was highest among the drilled and sealed mussels (80% higher than among the other treatments). The vulnerability of the population, the aim of the study and the duration of the experiment should decide what method to use for tissue sampling. For long-term experiments and repeated sampling, opening the mussels by prizing apart the valves is a better alternative than drilling holes in the shells, but depending on the morphology of the species it could be difficult to sample the anterior part of the mussel body. For a short experiment and to sample anterior parts, drilling the shells, leaving the holes open and using surgical forceps, seems to be an acceptable compromise between the different treatments used.