Richard K. Pipe

Contaminant induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study

Damage to lysosomes in isolated molluscan digestive cells was measured in vitro, following prior experimental in vivo exposure of mussels to the model polycyclic aromatic hydrocarbon fluoranthene for 7 days. Damage was assessed using the retention of the cationic diazine probe neutral red in the lysosomal compartment as a determinant of effect. The results showed that probe retention time was significantly reduced (P < 0.001) in the lysosomes of cells isolated from exposed mussels as compared to those from mussels exposed to the solvent vehicle alone. These findings indicate that the functional integrity of the lysosomal membrane is impaired following hydrocarbon exposure and further illustrates that lysosomes are the target of toxic action of pollutants. Corroborative evidence for fluoranthene-induced cell injury was apparent from increased accumulation of lipid and lysosomal hydrolase activity in tissue sections of digestive gland from the same experimental material.

Environmental contaminants influencing immunefunction in marine bivalve molluscs

The increased observation of pollution induced disease conditions in marine organisms has led to a growing interest on the effects of environmental contaminants on the immune system. Most studies on modulation of the immune system in bivalves by pollutants have concentrated on the effects of heavy metals and polycyclic aromatic hydrocarbons (PAHs). The current literature on contaminant effects on specific components of the bivalve immune system is reviewed together with the effects on susceptibility to infection. Data are presented showing the effects on immune parameters of exposure to Vibrio tubiashi following pre-exposure to copper or cadmium. Mussels exposed to cadmium for 7 days followed by 7 days exposure to V. tubiashi demonstrated significantly higher numbers of circulating haemocytes compared with non-Vibrio-exposed groups. Similar experiments conducted with copper exposure for both 7 days and 7 weeks followed by V. tubiashi for 7 days demonstrated a significant decrease in the percentage of circulating eosinophils compared with basophilic cells for both short and long term exposures. The intracellular release of superoxide (NBT reduction) by haemocytes was stimulated in Vibrio-challenged mussels with no copper pre-exposure but was significantly reduced in mussels pre-exposed to 0·2 ppm of copper for 7 weeks. The mortalities for the copper experiments showed increased levels with increasing copper concentration and were consistently higher in the V. tubiashi challenged mussels which had also been exposed to copper.

Copper induced immunomodulation in the marine mussel, Mytilus edulis

The effects on the immune response of the mussel, Mytilus edulis, of short-term, in-vivo exposure to copper were investigated under laboratory-controlled conditions. Parameters measured concentrated on the ability of the blood cells to destroy invading pathogens and included changes in the number and character of the circulating haemocytes, peroxidase and phenoloxidase enzyme activity in the blood cells, intra- and extracellular superoxide radical production, phagocytosis and uptake of neutral red. Copper concentrations of 0.02 and 0.05 ppm were found to increase significantly the total number of circulating haemocytes, while 0.2 and 0.5 ppm decreased the proportion of eosinophilic to basophilic cells. Intracellular superoxide production significantly decreased on exposure to 0.5 ppm copper, whereas phagocytic activity was stimulated at 0.2 ppm but not at 0.5 ppm. Copper exposures of 0.2 and 0.5 ppm reduced the percentage of haemocytes showing binding of lectins from Galanthus nivalis and Helix pomatia compared with haemocytes from mussels not dosed with copper. No significant alterations were found in peroxidase and phenoloxidase activity, binding of wheat germ agglutinin or uptake of neutral red. The results are discussed in the light of elucidating the possible relationship between environmental contaminants and increased disease susceptibility in aquatic organisms. The benefits of using a multi-assay approach to monitoring environmental pollution using such methods are highlighted.

The separation and characterisation of haemocytes from the mussel Mytilus edulis

Abstract.The separation of haemocytes from the mussel Mytilus edulis was carried out on continuous Percoll gradients. The haemocytes separated into three distinct layers, the first comprised 97% basophilic cells, the third comprised 84% eosinophilic cells and the middle layer was a mixture of eosinophilic and basophilic cells. Enzyme cytochemistry demonstrated arylsulphatase, phenol oxidase and peroxidase associated with the haemocytes from the third layer. Lectin-binding studies showed differential binding of lectins to the separated cells. The ultrastructural morphology demonstrated that the first layer of cells was composed predominantly of small agranular cells with a high nucleus to cytoplasm ratio. The second layer comprised a mixture of cells with the majority being granular cells with small granules. The third layer was almost exclusively composed of granular cells with small and large granules. Assays to assess the function of the different cells demonstrated that respiratory burst activity, measured as the reduction of cytochrome-c, was carried out almost entirely by the eosinophilic haemocytes. Similarly, levels of phagocytosis, measured as uptake of Escherichia coli, were much higher in the eosinophilic haemocytes. Of the potential mitogenic factors investigated, concanavalin A and pokeweed mitogen showed some evidence of inducing haemocyte proliferation.

Effects of fluoranthene on the immunocompetence of the common marine mussel, Mytilus edulis

The effects of a model polycyclic aromatic hydrocarbon on the immunocompetence of the marine mussel, Mytilus edulis, were demonstrated in laboratory-controlled experiments. Haemolymph from mussels, exposed for 7 days to three concentrations of fluoranthene, was extracted and assays carried out to investigate total and differential haemocyte numbers, peroxidase and phenol oxidase activity, production of reactive oxygen metabolites and release of lysosomal enzymes. Fluoranthene exposure at 200 and 400 μg l−1 resulted in an increase in the total number of circulating haemocytes but did not affect proportions of eosinophilic to basophilic blood cells. The release of reactive oxygen metabolites was also significantly stimulated. The percentage of haemocytes exhibiting peroxidase and phenol oxidase activity increased significantly at the highest contaminant concentration. The release during phagocytosis of chymotrypsin-like enzyme from the haemocytes was inhibited at all fluoranthene levels. The observed effects on immunocompetence of the mussel, on exposure to environmentally realistic levels of contaminant, demonstrate the necessity for further work in order to establish a relationship between pollutants and disease susceptibility. The variability in effects on different aspects of the immune response emphasises the need for a multi-assay approach to pollution monitoring.

Evidence for environmentally derived immunomodulation in mussels from the Venice Lagoon

Abstract Mussels, Mytilus galloprovincialis , were sampled at three times of year, from various sites within the Venice Lagoon and a reference Platform site in the north Adriatic. The immune response of the mussels was assessed using a range of assays which included total and differential cell counts, phagocytosis, degradative enzyme levels and release of reactive oxygen metabolites. Chlorinated hydrocarbons, including lindane, DDT and PCBs, together with trace metal levels were measured in digestive gland tissues from the mussels. The measurements of immune response and the contaminant levels showed seasonal fluctuations. However, the results did demonstrate significant differences in a number of immunocompetence assays which in some instances showed some correlation with the levels of contaminants measured in the tissues. In particular, total and differential cell counts, phagocytosis and release of reactive oxygen metabolites showed a degree of correlation with contaminant burdens although other factors were clearly involved. The results confirm the requirement for using a range of assays for monitoring pollution effects.

Comparisons of PAH-induced immunomodulation in three bivalve molluscs

There is growing evidence that contaminants may be partly responsible for the observed increase in disease in marine organisms by adversely affecting their immunity. Bivalve molluscs are common sentinels used in invertebrate immunotoxicology, however, to date, studies have been restricted to a few resilient species. This present study is a comparative investigation into the effects of the polycyclic aromatic hydrocarbon, phenanthrene, on the immunocompetence of three bivalve species. The commonly-studied marine mussel, Mytilus edulis, was compared with two species that have never been studied with respect to immunomodulation, namely, the edible cockle, Cerastoderma edule and the razor shell, Ensis siliqua. Animals were exposed to a range of phenanthrene concentrations (50, 100, 200 or 400 microg l(-1)) and haemocyte immune parameters, including haemocyte counts, phagocytosis, superoxide generation, lysosomal enzymes and lectin-binding, were monitored. Aims were not only to extend existing knowledge of bivalve immunotoxicology, but also to establish whether contaminant-induced immunomodulation in the sentinel species, M. edulis, is comparable to that observed in other bivalves. Results showed that the immune response of the three species was differentially affected by phenanthrene exposure, with immunomodulation in M. edulis not reflecting the immunological changes observed in the other two species. This suggests M. edulis may not be a suitable sentinel bivalve, and that other species, such as C. edule, may more accurately reflect the general immunological response of this group of marine animals.

Modulations in the immune defences of mussels (Mytilus edulis) from contaminated sites in the UK

To date, there have been only limited investigations of immunocompetence in natural invertebrate populations. The current study monitored a variety of immune parameters in the marine mussel, Mytilus edulis, from six sites within the UK, comparing ones with histories of severe contamination problems with relatively uncontaminated reference sites. Assays for immunocompetence in live blood cells (haemocytes) included tests for phagocytosis, superoxide generation and enzyme activity. Monoclonal antibody probes were also used to investigate variations in molecular characteristics of the haemocytes. The results showed that significant differences in immunocompetence were evident between mussels from contaminated and reference sites. Haemocytes of mussels from contaminated sites showed enhanced superoxide production and reduced activity of degradative enzymes, effects which may result in tissue damage or have implications for the efficient destruction of invading microorganisms. Significant reductions in the binding of certain monoclonal antibodies to haemocyte epitopes were observed too in mussels from contaminated sites. The results from this study show that immunomodulation is evident in animals from chronically contaminated sites, although it is difficult to attribute observed changes to any one contaminant. Certain modulations, such as increased superoxide generation, occurred in mussels from all three contaminated sites, while others were observed only at one. The results indicate that not all immune parameters are affected by contamination and that the type and extent of effects on immune defences vary with the nature or concentration of the contaminants.

Sub-populations of haemocytes in the adult and developing marine mussel, Mytilus edulis, identified by use of monoclonal antibodies

Abstract.Monoclonal antibodies specific for haemocyte sub-populations in the mussel, Mytilus edulis, were raised by use of separated basophilic and eosinophilic cell types as antigens. The antibodies could be broadly divided into 3 groups, reactive with sub-populations of (1) basophilic granular haemocytes, (2) basophilic granular and hyaline cells and (3) eosinophilic granular cells. Non-selective antibodies staining all haemocytes were also generated. The antibodies bound to epitopes of differing molecular masses and, at the ultrastructural level, reacted principally with the granules of the haemocyte sub-populations. The antibodies were used to investigate haemocyte function and ontogeny and to test reactivity with haemocytes from mussels subject to varying degrees of pollution stress. Five antibodies showed reactivity with cells from the trochophore and veliger larvae of M. edulis, indicating that epitopes on adult mussel hae-mocytes are also present at much earlier stages in the life history. Reactivity with the larval stages was most prevalent with non-selective antibodies and those selective for basophilic haemocytes. When mussels from different sites were examined, both immunocytochemistry and ELISA showed reduced expression of a 140 kDa epitope in the haemocytes of mussels subject to greater contaminant loads. These results show that the monoclonal antibodies of the present study are valuable both in tracing immune-cell development and in detecting molecular changes under conditions of stress.

The effects of copper on actin and fibronectin Organization in Mytilus galloprovincialls haemocytes

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.