Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha.

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.

Assessing microbial contamination and antibiotic resistant bacteria using zebra mussels (Dreissena polymorpha).

Aquatic pollution with faecal bacteria and subsequent consumption of contaminated water or food is a worldwide issue that causes severe health effects (e.g. meningitis, salmonellosis, dysentery). In addition, the excessive use of antibiotics in animal husbandry and human medicine has enhanced the selective pressure on pathogenic bacteria, further increasing human health risks and detrimental effects on natural microbial communities. This urges the need to monitor faecal contamination using a time-integrated approach, as grab water samples can miss pathogen peaks. We tested the ability of zebra mussels (Dreissena polymorpha) to take up and depurate faecal indicator bacteria such as Escherichia coli and intestinal enterococci. Furthermore, we quantified the frequency of antibiotic resistant bacteria in water and mussels both in controlled laboratory tests and under in situ conditions downstream of a sewage treatment plant (STP). Laboratory results show that bacterial indicators in mussels were 132 times higher than their concentration in water, and that mussels retained bacteria up to 2 days after pulse exposure. Field results show decreasing bacterial concentrations in both water and mussels downstream the STP, with maximum E. coli concentrations ranging 173-9 cfu mL-1 in water and 2970-330 cfu g-1 in mussels. Similarly, enterococci ranged 59-4 cfu mL-1 and 1450-240 cfu g-1 in water and mussels, respectively. High proportions of antibiotic resistant E. coli were found in mussels (72%) and water (65%), and slightly lower proportion of resistant enterococci was found in mussels (47%) and in water (34%). Moreover, 33% of the bacteria isolated from mussels were resistant to multiple antibiotics, which emphasizes that resistance is a common feature in surface waters and highlights the need for safe water management. Our results show that zebra mussels provide an efficient, time-integrating tool for quantifying faecal indicators, including resistant and multidrug resistant bacteria.

Removal of enteric viruses and Escherichia coli from municipal treated effluent by zebra mussels.

Dreissena polymorpha is a widespread filter-feeder species, resistant to a broad range of environmental conditions and different types of pollutants,which has recently colonized Italian freshwaters. Although widely used to monitor pollution in freshwater environments, this species is also an important food source for some fish and water birds. It can also be used to concentrate or remove particulate organic matter to interrupt avian-to-human transmission of pollutants and control health risks for animals and humans. In this study, the accumulation/inactivation in D. polymorpha of human health-related spiked enteric viruses was described. The removal of endogenous Escherichia coli, the classical indicator of fecal contamination,was tested as well.Our preliminary lab-scale results demonstrate that zebra mussels can reduce significantly poliovirus titer after 24 h and rotavirus titer after 8 h. E. coli counts were also reduced in the presence of zebra mussels by about 1.5 log after 4 h and nearly completely after 24 h. The fate of the two enteric viruses after concentration by zebra mussels was also investigated after mechanical disruption of the tissues. To our knowledge, the accumulation from water and inactivation of human health-related enteric viruses by zebra mussels has never been reported.

Bioaccumulation of pathogenic bacteria and amoeba by zebra mussels and their presence in watercourses.

Dreissena polymorpha (the zebra mussel) has been invading freshwater bodies in Europe since the beginning of the nineteenth century. Filter-feeding organisms can accumulate and concentrate both chemical and biological contaminants in their tissues. Therefore, zebra mussels are recognized as indicators of freshwater quality. In this work, the capacity of the zebra mussel to accumulate human pathogenic bacteria and protozoa has been evaluated and the sanitary risk associated with their presence in surface water has also been assessed. The results show a good correlation between the pathogenic bacteria concentration in zebra mussels and in watercourses. Zebra mussels could therefore be used as an indicator of biological contamination. The bacteria (Escherichia coli, Enterococcus spp., Pseudomonas spp., and Salmonella spp.) and parasites (Cryptosporidium oocysts and free-living amoebae) detected in these mussels reflect a potential sanitary risk in water.

Shell biofilm nitrification and gut denitrification contribute to emission of nitrous oxide by the invasive freshwater mussel Dreissena polymorpha (zebra mussel).

Nitrification in shell biofilms and denitrification in the gut of the animal accounted for N(2)O emission by Dreissena polymorpha (Bivalvia), as shown by gas chromatography and gene expression analysis. The mussel's ammonium excretion was sufficient to sustain N(2)O production and thus potentially uncouples invertebrate N(2)O production from environmental N concentrations.

Parasitism can be a confounding factor in assessing the response of zebra mussels to water contamination.

Biological responses measured in aquatic organisms to monitor environmental pollution could be also affected by different biotic and abiotic factors. Among these environmental factors, parasitism has often been neglected even if infection by parasites is very frequent. In the present field investigation, the parasite infra-communities and zebra mussel biological responses were studied up- and downstream a waste water treatment plant in northeast France. In both sites, mussels were infected by ciliates and/or intracellular bacteria, but prevalence rates and infection intensities were different according to the habitat. Concerning the biological responses differences were observed related to the site quality and the infection status. Parasitism affects both systems but seemed to depend mainly on environmental conditions. The influence of parasites is not constant, but remains important to consider it as a potential confounding factor in ecotoxicological studies. This study also emphasizes the interesting use of integrative indexes to synthesize data set.

Identification of the molecules involved in zebra mussel (Dreissena polymorpha) hemocytes host defense.

The invasion of North American waterbodies by the zebra mussel (Dreissena polymorpha) has caused ecological catastrophies in North America. Unfortunately, little is known about this nuisance mollusk and its host defense mechanisms. In this study, 32 expressed sequence tags (ESTs) associated with hemocyte stimulation were obtained from a suppression subtractive hybridization (SSH) cDNA library. This SSH-cDNA library was produced by using a cDNA library of naïve hemocytes as the driver and a cDNA library of hemocytes stimulated with a mixture of microbial antigens namely, lipopolysacchride (LPS), peptidoglycan (PGN), and zymosan (ZYM), as the tester. The driver cDNA was subtracted from the tester to increase the relative abundance of the cDNAs that were induced by stimulations. The putative function of 27 ESTs were obtained by using the homologue searching program BLASTx and BLASTn. Four ESTs encoding the protein product homologous to matrilin (Matrn, AM503947), heat shock protein 70 (HSP70, EU835391), seryl-tRNA synthetase (STS, AM503950), and glycine-rich protein (GRP, AM502279) were selected for a subsequent study using quantitative PCR (qPCR) assays with the RNA extracted from hemocytes stimulated with LPS alone. The results of quantitative PCR with stimulated hemocyte RNA demonstrated that the four candidate genes were upregulated by LPS stimulation. The expression levels of both HSP70 and Matrn genes between naïve and 1-h stimulated hemocyte samples are the most significant with 2.78 and 2.20 fold increases, respectively. The significant changes of GRP and STS genes were observed after 2-h stimulation. The phylogenetic analysis of HSP70 molecule indicated that this protein is phylogenetically close to the HSP70 identified from other mollusks. This study shed light on hemocyte-mediated host defense mechanisms of D. polymorpha.

A comparison of the benthic bacterial communities within and surrounding Dreissena clusters in lakes.

The impact of Dreissena (Dreissena polymorpha and D. bugensis) on the benthic bacterial community in lakes is largely unknown. Therefore, we quantified differences in the structure and activity of bacterial communities living in sediments (1) associated with Dreissena clusters, and (2) unassociated with established clusters (lake bottom sediments). Dreissena clusters and sediments were collected from locations in Lake Erie, Lake Ontario, and several inland lakes. Denaturing gradient gel electrophoresis (DGGE) analysis of the benthic bacterial community showed that the bacterial populations selected for by Dreissena represent a subset of the bottom communities and are geographically distinct. Community-level physiological profiling (CLPP) showed that overall bacterial activity and metabolic diversity were enhanced by the presence of clusters in all samples, with the exception of those harvested from the two Lake Erie sites. Therefore, Dreissena appears to affect both structure and metabolic function of the benthic bacterial community and may have yet unexplored ecosystem and food web consequences.

Pseudodiarrhoea in zebra mussels Dreissena polymorpha (Pallas) exposed to microcystins.

Microcystins are produced by bloom-forming cyanobacteria and pose significant health and ecological problems. In this study we show that zebra mussels respond differently to different strains of Microcystis aeruginosa, and that a highly toxic strain causes zebra mussels to produce large quantities of mucous pseudofaeces, 'pseudodiarrhoea', that are periodically expelled hydraulically through the pedal gape by shell valve adductions rather than by the normal ciliary tracts. Analysis of the pseudofaecal ejecta showed that the proportion of Microcystis aeruginosa relative to Asterionella formosa was high in the pseudofaeces and even higher in the 'pseudodiarrhoea' when a mixed diet was given to the mussels. This confirms that very toxic Microcystis aeruginosa were preferentially being rejected by comparison with the non-toxic diatom in the pseudofaeces and even more so in the 'pseudodiarrhoea'. Such selective rejection was not observed with low or non-toxic strains and would therefore tend to enhance the presence of toxic Microcystis aeruginosa in mixed Microcystis aeruginosa cyanobacterial blooms, as well as transferring toxins from the water column to the benthos. The observed acute irritant response to the toxin represents the first demonstration of an adverse sublethal effect of microcystins on invertebrate ecophysiology. Our results also suggest that it could be a specific response to microcystin-LF, a little studied toxin variant.