Inke Sunila

Acute histological responses of the gill of the mussel, Mytilus edulis, to exposure by environmental pollutants

Abstract Acute effects of the heavy metals cadmium, copper, lead, cobalt, iron, and silver, and the organic pollutants PCB, DDT, and dieldrin on the gill of the mussel Mytilus edulis were studied in aquaria in brackish water (7‰). An inflammatory reaction was evoked by an exposure to cadmium, copper, lead, and silver. The inflammatory reaction in the gill began with enlargement of the postlateral cells, after which the blood spaces dilated and granular hemocytes migrated to the epithelia. Histological changes differed after an exposure to different pollutants. Exposure to cobalt, iron, or dieldrin had no effect. Copper exposure caused endothelial edema and deformation of abfrontal parts of the filaments. Lead caused loss of lateral cilia or sloughing of lateral cells. After an exposure to silver, endothelial cells became vacuolated. Organic pollutants caused loosening of intercellular connections in the epithelia and shrinkage of the cells.

Chronic histopathological effects of short-term copper and cadmium exposure on the gill of the mussel, Mytilus edulis

Abstract Mussels, Mytilus edulis , were exposed to elevated concentrations of copper (0.1, 0.2, 0.4, and 0.8 mg/liter) and cadmium (1, 2, 4, and 8 mg/liter) in aquaria. After the 24 hr exposure they were put in cages submerged in the sea. Samples from the gills were taken over a period of 1 year for histopathological analysis. Acute changes occurring were the swelling of the endothelial cells and the detachment of the abfrontal cells in the copper-exposed gills, and the dilatation of the branchial veins in the cadmium-exposed gills. The gills were invaded by granulocytes, indicating an inflammatory reaction. After 1 year, cavities were formed under the proximal ends of the postlateral cells in the copper- and cadmium-exposed mussels. Copper-exposed mussels had low gills with folded edges. Forty-four percent of the copper-exposed gills had structural deformities caused by the fusion of the gill filaments. The abnormalities were of the following types: fusion of frontal parts of the filaments, fusion of abfrontal parts of the filaments, or interlamellar fusion of the abfrontal parts of the filaments of opposite lamella.

Combined effects of a parasite, QPX, and the harmful-alga, Prorocentrum minimum on northern quahogs, Mercenaria mercenaria

Northern quahogs, Mercenaria mercenaria (L.), frequently are infected with the parasite Quahog Parasite Unknown (QPX, Labyrintohomorpha, Thraustochytriales), which can cause morbidity and mortality of the quahogs. Possible interactions between this parasitic disease and exposure to the harmful dinoflagellate Prorocentrum minimum in M. mercenaria were studied experimentally. Quahogs from Massachusetts with variable intensity of QPX infection were exposed, under controlled laboratory conditions, to cultured P. minimum added to the natural plankton at a cell density equivalent to a natural bloom. After 5 days of exposure, individual clams were diagnosed histologically to assess prevalence and intensity of parasitic infection, as well as other pathological conditions. Further, cellular defense status of clams was evaluated by analyzing hemocyte parameters (morphological and functional) using flow-cytometry. Exposure of quahogs to P. minimum resulted in: a lower percentage of phagocytic hemocytes, higher production of reactive oxygen species (ROS), larger hemocyte size, more-numerous hemocytic aggregates, and increased numbers of hemocytes in gills accompanied by vacuolation and hyperplasia of the water-tubular epithelial cells of the gills. Quahogs had a low prevalence of QPX; by chance, the parasite was present only in quahogs exposed to P. minimum. Thus, the effect of QPX alone on the hemocyte parameters of quahogs could not be assessed in this experiment, but it was possible to assess different responses of infected versus non-infected quahogs to P. minimum. QPX-infected quahogs exposed to P. minimum had repressed percentage of phagocytic hemocytes, consistent with immuno-modulating effect of P. minimum upon several molluscan species, as well as smaller hemocytes and increased hemocyte infiltration throughout the soft tissues. This experiment demonstrates the importance of considering interactive effects of different factors on the immunology and histopathology of bivalve shellfish, and highlights the importance of considering the presence of parasites when bivalves are subjected to harmful-algal blooms.

Survival, growth and shell deformities of copper- and cadmium-exposed mussels (Mytilus edulis L.) in brackish water

Mussels, Mytilus edulis L., were exposed to elevated concentrations of copper or cadmium in the laboratory, then placed in cages in the sea (salinity 7‰). One year later maximum lengths of the mussels were measured and shells screened for deformities. Growth was 0·6 cm year−1 in the control cages and retarded in cages of exposed mussels. A total of 63% of cadmium-exposed and 46% of copper-exposed mussels had shell deformities. In the control cages 26% were deformed while in a natural population only 3% were deformed. The proportion of deformities to growth was inversely related. Low concentrations showed their injuriousness in this long-term test, probably due to the closing of the valves during exposure to high concentrations.

Studies on the acute toxicity of the antineoplastic metal chelate trans-bis(salicylaldoximato)copper(II) in rats

The acute toxicity of the recently discovered antiproliferative and antineoplastic agent trans-bis- (salicylaldoximato)copper(II) (CuSAO2) has been studied in Wistar rats. When given perorally as a suspension, the compound had no acute lethal toxicity even at the dose of 5000 mg per kg body weight. The peroral LD50 value (in rats) of the corresponding free ligand, salicylaldoxime, is known to be only 400 mg/kg. The decreased toxicity of CuSAO2, as compared to the free ligand, may at least in part be due to its lack of absorption. CuSAO2 was found to be absorbed poorly, if at all, when given perorally. When CuSAO2 was given intraperitoneally to Wistar rats, its absorption was found to be incomplete, and therefore no attempt was made to determine its intraperitoneal LD50 value.

Respiration of sarcoma cells from the soft-shell clam Mya arenaria L. under various conditions

Abstract Oxygen consumption of sarcoma cells from the soft-shell clam Mya arenaria L. was studied. Hemolymph of clams in the terminal stage of sarcoma was strongly hypoxic. In early cases of sarcoma, partial pressure of oxygen in the hemolymph did not differ from the control animals but declined as the sarcoma advanced, and finally fell below the detection limit causing death of sarcomatous clams. Mitotic index declined as the sarcoma progressed and was 100 × lower in hypoxic plasma in the terminal stage than in early cases. Oxygen consumption of the sarcoma cells in 15% salinity seawater (18 °C) was 3.9 × 10 −4 μ l· h −1 ·cell − . Respiration rates declined in salinities and > 15% . Temperature stress (37 °C) depressed oxygen consumption before mortality could be detected but returning the cells to 18 °C increased oxygen consumption above the initial level.

Physiological and pathological changes in the eastern oyster Crassostrea virginica infested with the trematode Bucephalus sp. and exposed to the toxic dinoflagellate Alexandrium fundyense.

Effects of experimental exposure to Alexandrium fundyense, a Paralytic Shellfish Toxin (PST) producer known to affect bivalve physiological condition, upon eastern oysters, Crassostrea virginica with a variable natural infestation of the digenetic trematode Bucephalus sp. were determined. After a three-week exposure to cultured A. fundyense or to a control algal treatment with a non-toxic dinoflagellate, adult oysters were assessed for a suite of variables: histopathological condition, hematological variables (total and differential hemocyte counts, morphology), hemocyte functions (Reactive Oxygen Species (ROS) production and mitochondrial membrane potential), and expression in gills of genes involved in immune responses and cellular protection (MnSOD, CAT, GPX, MT-IV, galectin CvGal) or suspected to be (Dominin, Segon). By comparing individual oysters infested heavily with Bucephalus sp. and uninfested individuals, we found altered gonad and digestive gland tissue and an inflammatory response (increased hemocyte concentration in circulating hemolymph and hemocyte infiltrations in tissues) associated with trematode infestation. Exposure to A. fundyense led to a higher weighted prevalence of infection by the protozoan parasite Perkinsus marinus, responsible for Dermo disease. Additionally, exposure to A. fundyense in trematode-infested oysters was associated with the highest prevalence of P. marinus infection. These observations suggest that the development of P. marinus infection was advanced by A. fundyense exposure, and that, in trematode-infested oysters, P. marinus risk of infection was higher when exposed to A. fundyense. These effects were associated with suppression of the inflammatory response to trematode infestation by A. fundyense exposure. Additionally, the combination of trematode infestation and A. fundyense exposure caused degeneration of adductor muscle fibers, suggesting alteration of valve movements and catch state, which could increase susceptibility to predation. Altogether, these results suggest that exposure of trematode-infested oysters to A. fundyense can lead to overall physiological weakness that decrease oyster defense mechanisms.

SEVERAL CASES OF DISSEMINATED NEOPLASIA IN MUSSELS MYTILUS EDULIS (L.) IN WESTERN LONG ISLAND SOUND

Abstract Eight cases of disseminated neoplasia were found among 540 specimens of Mytilus edulis collected from an intertidal beach in Connecticut in western Long Island Sound. This was unusual, because disseminated neoplasia is very rare in M. edulis but causes epizootic mortalities in another mussel species, M. trossulus. According to histology, mussels showed a continuum of disease progression, from early stages with a few anaplastic cells around the stomach epithelium to more advanced cases with multiple foci of neoplastic cells in the tissues, finally to terminal cases with profuse infiltration of neoplastic cells in all tissues. Flowcytometric hemocyte analyses were performed to compare immune functions between neoplastic and healthy mussels. Circulating hemocytes from the neoplastic mussels showed significantly less phagocytosis and significantly more apoptosis than hemocytes from the healthy mussels. Hemocyte cell density measured by flow cytometry increased in the hemolymph with progression of the disease on histological sections. In situ hybridization was performed to detect apoptosis also on paraffin sections. There were more apoptotic neoplastic cells in the early stages of the disease than in the later stages. These observations suggest the need for further studies on apoptosis-regulating genes to explain differences in susceptibility to neoplasia of different Mytilus species, and the role of apoptosis in the progression of disseminated neoplasia.

Pollution-related histopathological changes in the mussel mytilus edulis L. in the Baltic Sea

Abstract Mussels, in their marine habitat, are exposed to several environmental stressors, such as pollutants, oxygen deficiencies or infectious agents. In marine pollution monitoring, it is important to distinguish changes caused by pollutants from changes caused by other stressors. This paper describes pollution-related histopathological changes in mussels sampled from the Baltic Sea. Field samples were collected from 23 polluted and unpolluted sites from the coasts of Finland (salinity 4·5–7‰). Histopathological observations are surveyed against previous laboratory studies, where mussels were exposed in aquaria to copper or cadmium in brackish water. Changes, which were induced in aquaria and occurred also at polluted sites, were phagocytosis of sperm in the follicles and gill lesions (hyperplasia of epithelia, metaplastic interfilamentar junctions and a chronic inflammatory reaction). In addition, occurrence of inflammatory reactions, ulcers and hemorrhages of the digestive tract and kidney lesions correlated with polluted sites.

Immune response and mechanical stress susceptibility in diseased oysters, Crassostrea virginica

Eastern oysters, Crassostrea virginica, naturally infected with the parasite Perkinsus marinus were subjected to a mechanical stress by centrifugation, and immune parameters, pathological conditions, and gene expression of selected transcripts were compared to uninfected controls. Immune parameters were assessed by flow cytometry, pathology and parasites by histotechnology and fluid thioglycollate assays, and gene expression by quantitative RT-PCR. Irrespective of mechanical stress, an increased number of hemocytes were observed in P. marinus-infected oysters that corresponded to increased expression of genes that have been shown to be involved in inflammation and apoptosis, two processes associated with regulating immune cell populations. Mechanically stressed, diseased oysters showed histological gill abnormalities and aggregations of hemocytes in tissues not seen in stressed, uninfected oysters. Expression of a high-mobility group protein and hemocyte phagocytosis were significantly upregulated upon mechanical stress only in uninfected oysters. The results of this study demonstrate the role of inflammation in the oyster immune response including possible underlying molecular mechanisms. Furthermore, this study highlights the importance of considering mechanical stressors when characterizing oyster immune function.