A carbon and nitrogen flux model of mussel digestive gland epithelial cells and their simulated response to pollutants

The mussel digestive gland epithelial cells provide a key interface between the organism and pollutants such as aromatic hydrocarbons. The simulation of their uptake and export mechanisms as well as an internal protein degradation pathway, and any subsequent disruption to any of them, has been undertaken. A computational model is described, which simulates the flow of carbon and nitrogen through a mussel's digestive cell. The model uses a compartmentalised view of the cell with inviolate 'pipelines' connecting each of the volume-variable partitions. Only the major physiological pathways relevant to the flow of either carbon or nitrogen or volume are modelled. Simulated response to hydrocarbon exposure is examined.     

Environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status

The potential prognostic use of lysosomal reactions to environmental pollutants is explored in relation to predicting animal health in marine mussels, based on diagnostic biomarker data. Cellular lysosomes are already known to accumulate many metals and organic xenobiotics and the lysosomal accumulation of the carcinogenic polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) is demonstrated here in the hepatopancreatic digestive cells and ovarian oocytes of the blue mussel. Lysosomal membrane integrity or stability appears to be a generic indicator of cellular well-being in eukaryotes; and in bivalve molluscs it is correlated with total oxygen and nitrogen radical scavenging capacity (TOSC), protein synthesis, scope for growth and larval viability; and inversely correlated with DNA damage (micronuclei), as well as lysosomal swelling (volume density), lipidosis and lipofuscinosis, which are all characteristic of failed or incomplete autophagy. Integration of multiple biomarker data is achieved using multivariate statistics and then mapped onto "health status space" by using lysosomal membrane stability as a measure of cellular well-being. This is viewed as a crucial step towards the derivation of explanatory frameworks for prediction of pollutant impact on animal health; and has facilitated the development of a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells, tissues and the whole animal. This model has also complemented the creation and use of a cell-based bioenergetic computational model of molluscan hepatopancreatic cells that simulates lysosomal and cellular reactions to pollutants. More speculatively, the use of coupled empirical measurements of biomarker reactions and modelling is proposed as a practical approach to the development of an operational toolbox for predicting the health of the environment.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

Age-related differences in the recovery of lysosomes from stress-induced pathological reactions in marine mussels

This study was conducted to investigate the adaptability of marine mussels Mytilus edulis of increasing age to induced stress and subsequent recovery. Lysosomes, present in large numbers in the molluscan digestive gland, play a major role in intracellular digestion, and the stability of their membranes provides a sensitive biomarker for generalised cell injury which is correlated with the stress response of the whole animal. Lysosomal stability was measured in mussels of three age groups (2–4, 6–8 and ≥ 10 years) during exposure to hypoxia/hyperthermia and, in a separate experiment, to copper (50 ppb: where billion = 10⁹). The lysosomal reactions of all three age groups to both experimental stressors were similar. However, recovery from the induced pathological reactions was most pronounced in the youngest animals and least apparent in the oldest group. These findings indicate that the stress reaction is independent of age but that the potential for recovery of lysosomal integrity is age-related.     

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.     

DNA adducts and polycyclic aromatic hydrocarbon (PAH) tissue levels in blue mussels (Mytilus spp.) from Nordic coastal sites

DNA adducts in gills and digestive gland, as well as polycyclic aromatic hydrocarbon (PAH) tissue levels were analysed in blue mussels (Mytilus spp.) from Nordic coastal areas (Iceland, Norway and Sweden) with diffuse or point sources of PAHs of various origins. Both DNA adduct and PAH tissue levels were generally low, indicating low PAH exposure to the mussels in the areas studied. DNA adducts were found to be higher in gills than in digestive gland of the mussels at all sites studied. Elevated DNA adduct levels in gills were found at 6 sites out of 18 compared to reference sites in respective coastal zones. Adduct levels ranged from 0.5 to 10 nmol adducts/mol normal nucleotides, being highest in mussels from Reykjavík harbour, Iceland (intertidal mussels), and from Fiskaatangen, Norway (subtidal mussels). Total PAH tissue levels in the mussels ranged between 40 and 11,670 ng/g dry wt., and were significantly correlated with DNA adduct levels (r(2)=0.73, p<0.001). PAH ratio values indicated that the PAHs were in most cases of pyrolytic origin, but with petrogenic input near harbours and an oil refinery.     

Biomarkers and trace metals in the digestive gland of indigenous and transplanted mussels, Mytilus galloprovincialis, in Venice Lagoon, Italy.

The aim of this study was to investigate the cellular and biochemical response of mussels, Mytilus galloprovincialis, transplanted from a relatively pristine site to a polluted one and vice versa in the Lagoon of Venice (northeast of Italy) and to apply auto-metallography, a rapid and sensitive histochemical technique, to determine the bio-available fraction of heavy metals accumulated in the body tissues of organisms. Animal digestive glands have been used for morphological analyses (lysosomal volume, surface and numerical density, mean epithelial thickness, mean diverticular and luminar radius), autometallographical black silver deposits quantification and biochemical assays (superoxide dismutase and catalase activity). Furthermore, heavy metal content was determined by atomic absorption spectrophotometry (AAS) using standard procedures. The overall results indicate a direct influence of the environment in the thinning of the digestive cells and in the increasing number of lysosomes in mussels from the more polluted site. These data are in agreement with the metal content in digestive cell lysosomes as determined by autometallography, whereas AAS measurements show less significant differences.     

Role of metallothioneins in Cu and Cd accumulation and elimination in the g gill and digestive gland cells of mytilus galloprovincialis lam.

The data presented in this paper demonstrate that exposure of mussels to Cu (40 μg/liter) or Cd (200 μg/liter) for a period of 2 days is sufficient to induce Cu or Cd thioneins in the gills of the metal-exposed animals. The concentrations of Cu and Cd thioneins increase during the period of metal exposure (3–4 weeks), confirming the fact that metallothioneins play a fundamental role in the accumulation of these metals in the tissues examined (gills and digestive gland). Moreover, it has been demonstrated that when metal-loaded mussels are returned to the field for the recovery period, Cu is rapidly eliminated from the gill and digestive gland cells, showing a biological half-life of 9–10 days, whereas Cd is released much more slowly from the tissues, only about 50% of the total metal being lost after 4 months of detoxification. Interestingly enough, during the detoxification period the concentration of the metal bound to thioneins also follows the same pattern as the total metal present in the tissues, in fact the concentration of Cu thioneins decreases near the control level in about 24 days but, in contrast, the Cd-thionein concentration in the cytisol of the gill and digestive gland cells remaains high, decreasing only by about 40% at the end of the 4 month recovery period.The data presented also demonstrate that when Cd-loaded, detoxified mussels are exposed to Cu (100 μg/liter) for 6 h, the metal taken up by the gill cells is able to displace the Zn which is always present in the Cd-thionein fraction, but it does not, however, displace the Cd present. In this case also, period (20 days), whereas the Cd-thionein content decreases slightly. Such data seem to indicate that the metabolic characteristics of thioneins are closely related to the metal bound to them, particularly where the elimination rate of the metals from the cells is concerned.     

栉孔扇贝(Chlamys (Azumapecten) farreri) 消化盲囊的组织学和组织化学的研究

运用石蜡切片法、透射电镜技术及组织化学方法，对栉孔扇贝的消化盲囊进行了研究。结果表明消化盲囊的腺上皮由消化(吸收)细胞和分泌细胞组成，消化(吸收)细胞顶端有微绒毛，细胞内有酸性磷酸酶、碱性磷酸酶、脂酶和酯酶活性，还含有糖原和脂肪。是进行食物的细胞内消化和营养物质吸收的主要场所，并有储存能量的功能；分泌细胞内含粗面内质网、高尔基体和分泌颗粒，具有酸性磷酸酶和碱性磷酸酶活性。并含有丰富的RNA和蛋白质，有分泌消化酶的功能；消化盲囊导管上皮细胞顶端有纤毛和微绒毛，细胞内有酸性磷酸酶、碱性磷酸酶及脂酶活性，也能分泌消化酶。消化(吸收)细胞浅部的细胞质及其释放到盲囊腔中的胞质团中含铁；各部位不含钙。     

Evaluation of general and specific stress indices in mussels collected from populations subjected to different levels of heavy metal pollution

It has been shown that the rate of protein synthesis is significantly decreased in various tissues of mussels gathered from a coastal area, characterised by a high level of pollution, when compared with animals collected in the reference unpolluted area. Such a decrease may represent a general stress index. Additional biological parameters, such as amino acid uptake and RNA synthesis, are similarly decreased in the mussels collected from the polluted area.Moreover, it has been demonstrated that in the digestive gland of the animals sampled from the heavy metal polluted area the level of low molecular weight thionein-like Cu-binding proteins is three times higher than in the digestive gland of the control mussels. The increase in the level of metallothionein-like proteins in the digestive gland of the mussels appears to represent a specific stress index directly related to the presence in the sea water of high concentrations of heavy metals.     

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.     

菲律宾蛤仔消化系统的组织学和组织化学研究

用组织学和组织化学方法对菲律宾蛤仔的消化系统进行了研究。消化腺腺上皮由消化细胞和嗜碱性细胞组成。消化细胞呈现蛋白酶、非特异性酯酶、脂酶、酸性磷酸酶和碱性磷酸酶活性 ,表明能进行细胞内消化。嗜碱性细胞含丰富的RNA和蛋白质。消化道粘膜上皮由纤毛柱状细胞和少量的粘液细胞组成。晶杆囊基部与肠相连。肠纤毛柱状细胞呈现蛋白酶、非特异性酯酶、酸性磷酸酶和碱性磷酸酶活性。     

Use of the Comet assay to assess DNA damage in hemocytes and digestive gland cells of mussels and clams exposed to water contaminated with petroleum hydrocarbons

Oil spills can result in the deposition of large quantities of petroleum hydrocarbons into intertidal and shallow waters seriously impacting bivalve populations. Petroleum hydrocarbons are enriched in polycyclic aromatic hydrocarbons (PAH) and PAH analogs many of which may have potential to damage DNA. The Comet assay is useful for assessing DNA damage and has been used to a limited degree with aquatic organisms, but mostly with studies in vitro. We have carried out studies with the Comet assay to assess the DNA damaging potential of complex mixtures of petroleum hydrocarbons for bivalves. Experiments were carried out with mussels (Mytilus edulis) and clams (Mya arenaria) with dispersions and water soluble fractions of an Arabian crude oil which was also chemically characterized in detail by GC-MS. Pilot studies were first conducted to evaluate test performance and reproducibility. An interindividual coefficient of variation ranging from 17 to 30% was established for the assay with hemocytes and digestive gland cells of both species. Exposure to hydrocarbon fractions had no significant impact on clams. However, an increase in DNA damage was observed at P < 0.1 with digestive gland cells of mussels exposed to aqueous fractions of a light crude oil. These studies have demonstrated a potential for DNA damage in bivalves exposed to oil spills in inshore waters as well as potential for interspecies sensitivity.     

Histopathology of mussels (Mytilus sp.) from the Tamar estuary, UK

This study assessed numerous histological parameters as markers of health status in mussels (Mytilus spp.) collected from several locations along a contamination gradient on the River Tamar and two coastal sites on the north and south coast of Cornwall, UK. Twenty-seven health parameters were assessed including the presence of pathogens, inflammatory lesions, non-specific pathologies and reproductive condition. Logistic regression showed that reproductive condition and inflammatory lesions showed changes indicative of a contamination gradient response. Mussel populations further upstream within the Tamar were relatively less developed compared to mussels situated downstream in the estuary. Compared to coastal locations, mussels sampled within the Tamar also exhibited a higher prevalence of inflammatory lesions that increased at locations situated furthest upstream. Similarly, levels of lipofuscin within the kidney epithelium were highest in mussels sampled within the estuary and Whitsand Bay compared to Trebarwith Strand. The most prevalent pathogens observed during this survey included Rickettsia/Chlamydia-like organisms (R/CLO), gill ciliates including Ancistrum mytili, the copepod Mytilicola intestinalis, Ciliophora-like organisms and Marteilia sp. Marteilia sp. was observed in 31% of the mussels sampled from the Cremyll Ferry site. Generally, the range and prevalence of pathogens was highest from the two estuarine locations at Cremyll Ferry and Wilcove. No significant differences were observed between estuarine sampling sites and Whitsand Bay with respect to median epithelial cell height of digestive tubules, however mussels from Trebarwith Strand did exhibit a reduced median epithelial cell height compared to mussels sampled from other sampling sites. Species genotyping was also undertaken during this study in order to consider any histological differences in relation to species. Results are discussed in relation to histopathology health parameters and biological effects monitoring.     

Effets d'un effluent de fabrication du bioxyde de titane sur un mollusque (Mytilus edulis). Comparaison d'animaux traités expérimentalement et prélevés à proximité d'un rejet d'usine

The effects of an effluent from a factory producing titanium dioxide have been tested on mussels experimentally contaminated and collected in the proximity (2 miles) of the factory at Calais, France. In mussels contaminated for two weeks with the effluent (dilution in sea water: 1/500 to 1/10 000) neither mortality nor tissue lesions were observed. Chemical effects increased with the amounts of effluent: increased concentrations of Mn and Fe; storage of iron hydroxide, titanium oxide, vanadium, chromium, copper and, probably, aluminium, in the lysosomes of the digestive cells; extrusion of particles rich in Al, Ti; Cr, Fe and Zn in the basement membrane of the digestive tubules; contamination of the connective tissues with Cr, Fe and Cu; excretion of V, Cr and Mn by the kidney, increase in the renal excretion of Fe. The results show that two elements of the effluent (Al and Ti) are not assimilable, four elements (V, Cr, Mn and Cu) are assimilable, whereas iron is present in both forms. Mussels collected in the proximity of the factory exhibit none of the typical features of the contaminated animals. In addition, the results are inconsistent with recent data dealing with the occurrence of uranium, which could originate from the effluent, in mussels collected from the Pasde-Calais.     

Development of two novel CYP-antibodies and their use in a PCB exposure experiment with Mytilus edulis

In an attempt to learn more about the cytochrome P450 (CYP) system of mussels, we used protein databases and alignment software to extract highly conserved CYP sequences. From these alignments synthetic peptides were produced and used for rabbit immunisation, which yielded polyclonal antibodies against the CYP families 2 and 4. The antibodies were evaluated with Western Blot and ELISA assays, using digestive gland microsomal samples from the mussel Mytilus edulis. Western Blots revealed immunoreactions for both antibodies. The anti-CYP2 sequence rendered one major immunopositive protein of approximately 49 kDa size, and weak signals for proteins of approximately 41 and 56 kDa size. The anti-CYP4 sequence rendered two major bands of approximately 56 and 59 kDa size, and also a weak immunoreaction with a protein of approximately 43 kDa size. ELISA rendered only weak signals even with a 1:50 dilution of IgG-purified serum. A 10-day exposure to Aroclor 1254 did not appear to affect any of the immunopositive proteins, while total PCBs in soft bodies increased from 14-40 ng/g DW in controls to 373-638 ng/g DW in exposed mussels.     

Effects of linear alkylbenzene sulphonate (LAS) and cadmium in the digestive gland of mussel, Mytilus sp.

A laboratory study was carried out exposing mussels (Mytilus sp.) to linear alkylbenzene sulphonate (LAS) (6 mg litre⁻¹), Cd (0.05 mg litre⁻¹) and LAS plus Cd at the same concentrations. The aim was to assess the use of several histopathological and biochemical indices as potential biomarkers of the impact of these xenobiotics in the digestive gland of molluscs. Treated mussels actively accumulated Cd in the digestive gland compared with controls (p 0.01), the highest levels occurring after 30 days of exposure in the group treated with Cd plus LAS. Among several histological alterations screened in digestive gland tissues the thickness of digestive tubules in Cd treated animals decreased more markedly (p 0.01) than in LAS exposed mussels. As for biochemical parameters, the investigated antioxidant enzyme activities, superoxide dismutase (SOD), catalase, DT-diaphorase and glutathione peroxidase (GPX) did not show any significant induction due to these xenobiotics. However, a slight decrease of the antioxidant defences of the animals was detected after 30 days of exposure to contaminants.     

Does ecological redundancy maintain functioning of marine benthos on centennial to millennial time scales?

Predicting the ability of the biosphere to continue to deliver ecosystem services in the face of biodiversity loss and environmental change is a major challenge. The results of short‐term and small‐scale experimental studies are both equivocal and difficult to extrapolate from. In this study we use data on benthic palaeocommunities covering 4,000,000 years (in the Late Jurassic when temperate coastal seas in NW Europe experienced fluctuations in oxygenation). The biological traits associated with each species in the palaeocommunities were combined to index the delivery of ecological functions. Five ecosystem functions were examined: food for large mobile predators, biogenic habitat provision, nutrient recycling/regeneration, inorganic carbon sequestration and food‐web dynamics. In modern systems these ecological functions underpin ecosystem services that are important for human well‐being. Our results show that the supply of food for higher predators was remarkably constant during the 4,000,000 years, suggesting that redundancy amongst species in the assemblage drives the biodiversity–ecosystem function (BEF) relationship. By contrast, the provision of biogenic habitat varied with the occurrence of a relatively few taxa, a pattern consistent with a rivet type model of BEF. For nutrient regeneration, carbon sequestration and food‐web dynamics the patterns were complex and suggestive of an idiosyncratic model of BEF. To our knowledge this is the first study to quantify ecological functioning through deep time and demonstrates the utility of this approach to understanding long‐term patterns of BEF in both ancient and contemporary marine ecosystems. The delivery of all five ecological functions studied became increasingly variable as the regional climate became drier, thus modifying the supply of terrigenous nutrient inputs.     

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.     

Seston available as a food resource for the ribbed mussel (Geukensia demissa) in a North American,mid-Atlantic saltmarsh

We studied the composition of the <25 μm seston size fraction as a food resource potentially available to suspension feeding ribbed mussels, Geukensia demissa, over an annual cycle in Canary Creek saltmarsh, Delaware Bay. There were significant seasonal variations in the concentration of particulate organic carbon (POC), particulate organic nitrogen (PON), and total carbohydrate, but not cellulose. The concentration of cellulose, measured by hydrolytic cellulase enzyme assay, was relatively low (seasonal range 24 to 35 μg l⁻¹) and only comprised from 3% of total carbohydrate in May 1996 to 13% in November 1995. We used the biomass of microalgae, estimated from chlorophyll a, and abundance of free-living bacteria and heterotrophic nanoflagellates to calculate each component's equivalent carbon content. Microalgae were the most dominant carbon source (62% annually) among the four identified components (phytoplankton, bacteria, heterotrophic nanoflagellates, and cellulose) in all seasons except in August 1995 when carbon from bacteria was most abundant (55%). The annual average carbon equivalents of heterotrophic nanoflagellates and cellulose were relatively small (2 and 4%, respectively). The total concentration of POC in the seston was much greater than the carbon derived from the four identified components. The proportions that these identified components contributed to POC varied seasonally and combined only accounted for 8–24% of POC. Based on these estimates, the bulk of the POC in Canary Creek marsh was not associated with any of the four components we identified. We suggest that this uncharacterized material was some type of non-lignocellulosic, amorphous detritus of unknown utility as a food resource for ribbed mussels.