Detection of DNA damage in haemocytes of zebra mussel using comet assay

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 μg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 μg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.     

Effects of salinity on solute clearance from the freshwater bivalve, Dreissena polymorpha Pallas

Clearance of polyethylene glycol (PEG), inulin, or dextran that had been injected into the hemolymph of the mussel, Dreissena polymorpha, was measured in animals acclimated to pondwater (PW) or 10% seawater (SW). In addition, we measured the clearance of PEG from mussels acutely transferred into 10% SW and following return to PW after acclimation to 10% SW. Clearance values calculated for PW-acclimated mussels ranged from 2.0 to 3.3 ml (g dry tissue ċ h)^-1 and declined to 0.28 ml (g dry tissue ċ h)^-1 in 10% SW-acclimated animals. Transferring mussels into 10% SW resulted in a reduction in PEG clearance from the blood, coincident with the reduction of osmotic gradient. When 10% SW-acclimated mussels were returned to PW the clearance of PEG increased to rates observed in PW-acclimated animals within 1 h. The PEG clearance remained constant during the re-acclimation to PW even though the osmotic gradient declined from about 100 to 30 mosmol kg^-1. Clearance of the solutes used in this study was likely to be a measurement of renal filtration rate. The clearance values appeared to be maximal when the animals were in PW. The limited capacity to increase clearance in the face of an osmotic challenge may be a critical factor in restricting D. polymorpha to freshwater or lower salinity environments with small ranges in salinity.     

Development of an in vitro culture method for cells and tissues from the zebra mussel (<Emphasis Type="Italic">Dreissena polymorpha</Emphasis>)

Despite the successful transfer of mammalian in vitro techniques for use with fish and other vertebrates, little progress has been made in the area of invertebrate tissue culture. This paper describes the development of an in vitro technique for the culture of both cells in suspension and tissue explants from the gill, digestive gland and mantle of the zebra mussel (Dreissena polymorpha) and their successful maintenance in culture for up to 14 days. Cell suspensions from the gills and digestive gland were the most successful technique developed with viability >80% maintained for up to 8 days in culture, suitable for use in short term toxicity tests. Tissue explants from the mantle were also maintained in culture for up to 14 days. This paper describes the challenges involved in the development of a novel in vitro culture technique for aquatic invertebrates.     

Distribution and aspects of population dynamics of the zebra mussel,Dreissena polymorpha (Pallas, 1771), in the lake IJsselmeer area (The Netherlands)

Summary The distribution, settling and growth of the zebra mussel, Dreissena polymorpha Pallas, in Lake IJsselmeer and Lake Markermeer in The Netherlands were studied from 1980 to 1985. In these lakes D. polymorpha is the most important food source for wintering diving ducks. The study was part of an investigation into the carrying capacity of the lakes for these birds.     

Current and modeled potential distribution of the zebra mussel (Dreissena polymorpha) in Sweden

In this study, we report the results from a recently performed survey of zebra mussel in Sweden and present a risk model for the potential distribution of the species. The zebra mussel was found in 11 of the 30 surveyed lakes and rivers. Most of the observations were made in the north-eastern parts of Lake M?laren, i.e., in the basins Ekoln, Skarven, and G?rv?ln. Hierarchical partitioning and stepwise selection of water chemistry variables in a multiple logistic model identified pH and magnesium concentrations as the best predictors for zebra mussel occurrence. We conjecture that magnesium is an important predictor due to its crucial role in mussel physiology. The logistic model was applied on 2,781 lakes and in total 109 lakes (3.9%), most of them situated in specific areas in central Sweden, in the very south and on the island of Gotland, were predicted to be potentially at risk for zebra mussel invasions. The lakes potentially at risk for zebra mussel invasions are separated by the soft-water boreal lakes that constitute the vast majority of lakes on the Fennoscandian peninsula, thus forming barriers for further dispersal.     

Functional expression of human P-glycoprotein in Schizosaccharomyces pombe

Human MDR1 cDNA was introduced into the human cultured cells KB-3-1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressing human P-glycoprotein, was examined. KB-G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP-hydrolysis-deficient mutant did not confer KN3 resistance to these antibiotics. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation. The N-glycosylation-deficient mutant, however, conferred a typical MDR phenotype on KB-3-1. These results suggest that human P-glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.     

Social facilitation of male song by male and female conspecifics in the zebra finch, Taeniopygia guttata

Zebra finches are a ubiquitous model system for the study of vocal learning in animal communication. Their song has been well described, but its possible function(s) in social communication are only partly understood. The so-called ‘directed song’ is a high-intensity, high-performance song given during courtship in close proximity to the female, which is known to mediate mate choice and mating. However, this singing mode constitutes only a fraction of zebra finch males’ prolific song output. Potential communicative functions of their second, ‘undirected’ singing mode remain unresolved in the face of contradicting reports of both facilitating and inhibiting effects of social company on singing. We addressed this issue by experimentally manipulating social contexts in a within-subject design, comparing a solo versus male or female only company condition, each lasting for 24 h. Males’ total song output was significantly higher when a conspecific was in audible and visible distance than when they were alone. Male and female company had an equally facilitating effect on song output. Our findings thus indicate that singing motivation is facilitated rather than inhibited by social company, suggesting that singing in zebra finches might function both in inter- and intrasexual communication.     

Effect of temperature and salinity on acetylcholinesterase activity, a common pollution biomarker, in Mytilus sp. from the south-western Baltic Sea

Acetylcholinesterase (AChE) activity is a well established biomarker to monitor environmental pollution caused by neurotoxic compounds, such as organophosphorus and carbamate pesticides. The presence of these compounds results in a measurable inhibition of the enzyme. This has been shown for different marine species including blue mussels. Besides pollution, environmental variables may also have a direct or indirect effect on AChE activity, particularly in estuarine and brackish water environments. To assess the impact of abiotic factors on the AChE activity the seasonal course of gill AChE activity was followed in relation to temperature and salinity in Mytilus sp. collected from the south-western Baltic Sea. In addition, the effect of salinity on AChE activity was investigated in an experimental study. The AChE activity showed significant seasonal differences with maximum activities during the summer period and minimum activities in winter. These changes correlate significantly (p<0.001) and positively with water temperature. The experimental exposure of Mytilus sp. to different salinities (5, 7, 16 or 20 psu) resulted in changes in the gill AChE activity. Empirical orthogonal function (EOF) analysis revealed that AChE activity was significantly and negatively correlated with salinity (p<0.01). These results clearly demonstrate the need to consider the potential influence of temperature and salinity on AChE activity when applying this biomarker to monitor exposure to and effect of neurotoxic substances in estuarine and brackish water blue mussels.     

Influence of abiotic factors on bacterial proliferation and anoxic survival of the sea mussel Mytilus edulis L.

The effect of several abiotic factors (salinity, temperature and pH) on bacterial proliferation and survival time of the sea mussel Mytilus edulis L. were studied under anoxic incubations. In addition, the presence in the incubation media of ammonium and the volatile fatty acids propionate and acetate, both excreted fermentation products of the bivalve, was tested.Anoxic incubations with seawater diluted with demineralised water showed at the lowest salinity (50% seawater, SW) a significant increase in the capacity of M. edulis to survive anoxia as compared to both 75% SW and control [100% SW, corresponding to 32 practical salinity units (psu)]. Formation of biotic sulphide and ammonium occurred in all incubations. However, bacterial proliferation was postponed by 2-3 days at lowest salinity and accordingly, concentrations of both compounds were lower. Anoxic survival profiles of mussels collected from different habitats in the Dutch Scheldt area, characterised by differences in salinity (range from 17 to 31 psu), corresponded with the above salinity effect. Walsoorden mussels (17 psu) showed the longest (P<0.001) survival time under anoxia (LT₅₀=17.2 days) as compared with Paulina (27 psu) and Wemeldinge (31 psu) mussels (LT₅₀=12.8 and 9.8 days, respectively). Condition index (ratio of soft body weight to shell volume) was not correlated with anoxic survival time in untreated mussels, although this was clearly the case when the antibiotic chloramphenicol was added to the anoxic seawater.Acidification of the anoxic incubation medium had a positive effect on survival time. LT₅₀ values significantly (P<0.001) increased from 10.2 days at pH 8.1 to 11.6 and 11.5 days at pH 7.3 and 6.5, respectively. Biotic sulphide and ammonium accumulation as well as bacterial numbers were significantly lower at pH 7.3 and 6.5 as compared with pH 8.1. Anoxic incubations at 10 °C (LT₅₀=12.0 days) strongly increased survival time as compared to 18 °C (LT₅₀=5.9 days). The benefit of antibiotic addition was also stronger at lower temperature (10 °C).Addition of both propionate and acetate (0.5 mM) displayed no effect on mortality of mussels under anoxia, but ammonium (0.5 mM) caused a negative effect (P<0.001). Biotic sulphide and ammonium concentrations measured in both volatile fatty acid incubations were lower than the control situation, as well as total bacterial numbers.This study shows that environmental factors play a significant role in determining the course of bacterial infection and death of bivalves exposed to anoxia.     

Indigenous microflora and opportunistic pathogens of the freshwater zebra mussel, Dreissena polymorpha

Freshwater fouling invertebrate zebra mussels (Dreissena polymorpha) harbor a diverse population of microorganisms in the Great Lakes of North America. Among the indigenous microorganisms, selective species are opportunistic pathogens to zebra mussels. Pathogenicity to zebra mussels by opportunistic bacteria isolated from the mussels was investigated in this study. Among the more than 30 bacteria isolated from temperature-stressed mussels, Aeromonas media, A. veronii, A. salmonicida subsp. salmonicida, and Shewanella putrefaciens are virulent pathogens to juvenile zebra mussels. Inoculation of a bacterial concentration of A. media, A. salmonicida subsp. salmonicida and S. putrefaciens at 10⁷ cells per zebra mussel resulted in 100% mortality within 5 days, and only 64.9% for A. veronii. In contrast, mortality was less than 12.3% following inoculation of a sterile phosphate buffer solution as a control. In addition, mortality was dependent on the size of the pathogen population used in inoculation and the incubation temperature, indicating the close relationship between the bacterial population and subsequent death. On the mussel tissue, a dense microbial population was evident from the moribund mussels viewed with Scanning Electron Microscope (SEM). Opportunistic bacteria invaded and destroyed the D. polymorpha tissue after 7 days of incubation when the bacterial inoculation was larger than 10⁵ per zebra mussel. Our results suggest that mussels are reservoirs of opportunistic pathogenic microorganisms to aquatic organisms and humans and a better understanding of the microbial ecology of the mussels will provide insights to the possible health hazards from these microorganisms.     

The effect of seasonal variation in selective feeding by zebra mussels (Dreissena polymorpha) on phytoplankton community composition

1. To investigate the impact of zebra mussels ( Dreissena polymorpha ) on phytoplankton community composition, temporal variability in selective feeding by the mussels was determined from April to November 2005 in a natural lake using Delayed Fluorescence (DF) excitation spectroscopy.
2. Selective grazing by zebra mussels varied in relation to seasonal phytoplankton dynamics; mussels showed a consistent preference for cryptophytes and avoidance of chlorophytes and cyanobacteria. Diatoms, chrysophytes and dinoflagellates responded differentially to zebra mussel grazing depending on their size. Analysis of excreted products of the zebra mussels revealed that in addition to chlorophytes and cyanobacteria, phytoplankton >50  μ m and very small phytoplankton (≤7  μ m) were largely expelled in pseudofaeces.
3. The zebra mussel is a selective filter-feeder that alters its feeding behaviour in relation to phytoplankton composition to capture and ingest high quality phytoplankton, especially when phytoplankton occur in preferred size ranges. Flexibility of zebra mussel feeding behaviour and variation in susceptibility among phytoplankton groups to mussel ingestion indicate that invading zebra mussels could alter phytoplankton community composition of lakes and have important ecosystem consequences.     

Mechanisms and impact of differential fouling of the zebra mussel Dreissena polymorpha on different unionid bivalves

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Overexpression of P-glycoprotein in heat-and/or drug-resistant hepatoma variants

We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68.Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 g/ml colchicine (termed ashighly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stableheat-resistant variants of clone 2 were also isolated, which showed increaseddrug resistance to several drugs, i.e. they becamemoderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heatresistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against enviromental stress such as heat.Abbreviations hsp heat-shock protein - MDR multidrug resistance - P-gp P-glycoprotein     

Trinucleotide microsatellite loci for the zebra mussel Dreissena polymorpha,an invasive species in Europe and North America

The high mutation rate at microsatellite loci can supply important demographic information on founder events and range expansion in an invasive species such as the zebra mussel Dreissena polymorpha, following its initial introduction. In order to facilitate studies into the colonization patterns of this species in new habitats in Europe and North America, five trinucleotide microsatellite loci were isolated from a partial DNA library. Allelic diversity at all described loci was high, ranging from 20 to 35 alleles per locus. Homologous loci were not amplified in a second related invasive species, Dreisenna bugensis, using the primers developed here.     

Biology of the exotic zebra mussel,Dreissena polymorpha,in relation to native bivalves and its potential impact in Lake St. Clair

The zebra mussel, Dreissena polymorpha, is a new exotic species that was introduced into the Great Lakes as early as the fall of 1985. It differs markedly from native species of bivalves in its: (i) shell form; (ii) mode of life; (iii) reproductive potential; (iv) larval life cycle; (v) population dynamics; (vi) distribution, (vii) dispersal mechanisms; (viii) physiology; (ix) potential impact on the ecosystem; and (x) impact on society and the economy. In body form, it has an anterior umbone, a flat ventral surface with permanent aperature for the byssal apparatus and a shape that together make the animal well adapted for life on a hard surface. The shell has a zebra-stripe pattern, a heteromyarian muscle condition and lacks hinge teeth which make it easily identifiable from native bivalves. The zebra mussel is strongly byssate and has an epifaunal mode of life not seen in native bivalves. The species is dioecious and has external fertilization, the eggs developing into pelagic veligers which remains planktonic for approximately 4 weeks. Gametogenesis begins in late winter to early spring, veligers appear in the water column in late May to early June and disappear in mid to late October in Lake St. Clair. Adults live for about 2 years and have very rapid growth rates. Maximum shell lengths average 2.3 to 2.5 cm. Standing crops as high as 200 000 m^-2 are present in the 1-m depths of the Ontario shores. Infestations may be interfering with the normal metabolism of native unionid clams and there is potential of the unionid clam populations being reduced or even eliminated from Lake St. Clair.     

The pericardial glands of the zebra mussel: Ultrastructure and implication in lead detoxication process

Summary— Ultrastructural and microanalytical investigations of the pericardial gland of the freshwater mussel, Dreissena polymorpha have been performed to investigate the possible functional role of this organ in the detoxication process of lead. The cell-type of this organ exhibits the feature characteristics of podocytes, ie the typical pedicel-basal lamina complex and the well developed lysosomalvacuolar system. X-ray microanalysis demonstrated that large electron-dense granules referred to as lysosomes are the main target organelles in these cells to accumulate and sequestrate lead where the metal was associated with phosphorus and sulphur. Consequently, the pericardial gland plays an important role in the detoxication process and allows the organism to tolerate high lead concentration without suffering severe cell injury.     

Amplified fragment length polymorphism (AFLP) analysis of the genetic structure of the zebra mussel, Dreissena polymorpha, in the Mississippi River

1. We predicted that zebra mussel, Dreissena polymorpha (Pallas), genetic structure in the Mississippi River would follow a model of invasive species genetics, which predicts low genetic structure among populations of recently established species. This prediction was upheld in our previous genetic study using allozymes, however, one locus yielded anomalous results. 2. We employed amplified fragment length polymorphism (AFLP) analysis as a neutral marker to assess the amount of genetic structure within and among populations, and as a test of expected population structure from both invasion genetic theory, and the results from our previous study. 3. There was greater spatial differentiation, as measured by F_st, observed using AFLP's than for allozymes (P < 0.001). There was no evidence that AFLP variation conformed to an isolation by distance model, and genetic relationships of populations, as measured by AFLP markers, were not similar to those detected in our allozyme survey. 4. The lack of concordance between these two genetic marker systems probably reflects their differential responses to drift, migration, and selection occurring during this rapid invasion. Strong population structure is counter to predictions that populations of invasive species will not be differentiated, as with observations based on allozyme markers. Therefore, newly established species may require genetic surveys using multiple marker systems to evaluate population structure.     

Detection of a P-glycoprotein related pump in Chironomus larvae and its inhibition by verapamil and cyclosporin A

A membrane associated ATP-dependent efflux pump, similar in function to mammalian P-glycoprotein, was detected in anal papillae of Chironomus riparius larvae. Immunohistochemical analysis of larval tissues, using monoclonal antibodies against P-glycoprotein, was supplemented by functional in vivo and in vitro assays which confirmed the existence of a mechanism for transporting xenobiotic substances. The in vitro ATPase activity of homogenate fractions increased in the presence of typical P-glycoprotein substrates (vinblastine, actinomycin D or ivermectin). This increase was unaffected by inhibitors of other membrane ATPases (sodium azide, EGTA, ouabain), but sensitive to vanadate, cyclosporin A and verapamil which inhibit mammalian P-glycoprotein mediated ATP-consumption. Sublethal concentrations of specific P-glycoprotein-inhibitors such as verapamil or cyclosporin A synergistically enhanced the mortality of C. riparius towards ivermectin. Although cyclosporin A originates from entomopathogenic fungi, its mode of action in insects and its function during infection are not understood. Our results lend some credit to the hypothesis that this compound is possibly released to promote poisoning of the infected host by xenobiotics which are normally removed by a P-glycoprotein related pump. The putative role of insect P-glycoprotein homologues in the context of multiple resistance towards insecticides is discussed.     

Induction of autoantibodies to murine P-glycoprotein: consequences on drug sensitivity in MDR cancer cells and on the expression of mdr genes in organs

Overexpression of the 170 kDa plasma membrane P-glycoprotein (P-gp) represents the most common MDR mechanism in chemotherapy. In this work, specific autoantibodies to fragments from extracellular loops 1, 2, and 4 of the murine MDR1 P-gp were elicited in mice using synthetic palmitoylated peptides reconstituted in liposomes and alum. The highest IgG level was observed after the third immunization and the immune response against lipopeptides was still detected more than 200 days after immunizations. Immunocytochemichal studies revealed that these antibodies were specific for P-gp. When incubated with P-gp-expressing MDR cell lines, serum from immunized mice restored sensitivity to either doxorubicin or vinblastine, or had no effect in a cell type specific manner, suggesting that several mechanisms may occur in the establishment of the MDR phenotype. The expression of mdr1 and mdr3 genes was unchanged in organs from mice immunized with palmitoylpeptides grafted on liposomes. These results suggest that the induction of autoantibodies to P-gp is a safe strategy to overcome MDR in cancer chemotherapy.     

The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7–7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.