Comparison of viability and phagocytic responses of hemocytes withdrawn from the bivalves Mytilus edulis and Dreissena polymorpha, and exposed to human parasitic protozoa.

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

Neuronal Development in the Larvae of the Invasive Biofouler Dreissena polymorpha (Mollusca: Bivalvia), with Special Attention to Sensory Elements and Swimming Behavior.

Although understanding of the neuronal development of Trochozoa has progressed recently, little attention has been paid to freshwater bivalves, including species with a strong ecological impact, such as the zebra mussel (Dreissena polymorpha). Therefore, an important question might concern how the developing nervous system is involved in the formation of the rapid and successful invasive behavior of this species. Our aim was to reveal the neuronal development of trochophore and veliger larvae of Dreissena, with special attention to the organization of sensory structures and their possible involvement in detecting environmental cues. After applying serotonin and FMRFamide immunocytochemistry, the first serotonin immunoreactive sensory elements appeared 16-18 hours after fertilization, whereas the first FMRFamide immunoreactive sensory cell was seen only at 32 hours of development (trochophore stage). Later, sensory elements were found in three parts of the larval body, including the apical organ, the posterior region, and the stomach. Although differences in the timing of appearance and the morphology of cells were observed, the two signaling systems showed basic similarity in their organization pattern until the end of the veliger stage. Pharmacological, physiological, and quantitative immunocytochemical investigations were also performed, suggesting the involvement of both the serotoninergic system and the FMRFamidergic system in sensomotor processes. Manipulation of the serotonin synthesis by para-chloroplenylalanine and 5-hydroxytryptophane, as well as application of increased salinity, influenced larval swimming activity, both accompanied by changes in immunofluorescence intensity. We concluded that these two early sensory systems may play an important role in the development of settlement competency of this biofouling invasive bivalve, Dreissena.

Nanoparticulate versus ionic silver: Behavior in the tank water, bioaccumulation, elimination and subcellular distribution in the freshwater mussel Dreissena polymorpha.

Zebra mussels (Dreissena polymorpha) were exposed to polyvinylpyrrolidone (PVP)-coated silver nanoparticles (AgNP; hydrodynamic diameter 80 nm; solid diameter 50 nm) to investigate the behavior of Ag in the tank water with respect to its uptake, bioaccumulation, elimination and subcellular distribution in the mussel soft tissue. Parallel experiments were performed with ionic Ag (AgNO3) to unravel possible differences between the metal forms. The recovery of the applied Ag concentration (500 µg/L) in the tank water was clearly affected by the metal source (AgNP < AgNO3) and water type (reconstituted water < tap water). Filtration (<0.45 µm) of water samples showed different effects on the quantified metal concentration depending on the water type and Ag form. Ag accumulation in the mussel soft tissue was neither influenced by the metal source nor by the water type. Ag concentrations in the mussel soft tissue did not decrease during 14 days of depuration. For both metal forms the Ag distribution within different subcellular fractions, i.e. metal-rich granules (MRG), cellular debris, organelles, heat-sensitive proteins (HSP) and metallothionein-like proteins (MTLP), revealed time-dependent changes which can be referred to intracellular Ag translocation processes. The results provide clear evidence for the uptake of Ag by the mussel soft tissue in nanoparticulate as well as in ionic form. Thus, zebra mussels could be used as effective accumulation indicators for environmental monitoring of both Ag forms.

Functional features of hemocyte subpopulations of the invasive mollusk species Dreissena polymorpha.

Dreissena polymorpha is a mussel species that invaded many lotic and lentic inland waters in Western Europe and North America. Its positive or negative interactions with biotic and abiotic components of ecosystems are numerous, making this bivalve the subject of numerous studies in ecology, ecophysiology and ecotoxicology. In these contexts, the functional characterization of the zebra mussel hemocytes is of particular interest, as hemocytes are central cells involved in vital functions (immunity, growth, reproduction) of molluscan physiology. Dreissena polymorpha circulating hemocytes populations were characterized by a combination of structural and functional analysis. Assessments were performed during two contrasted physiological periods for mussels (gametogenesis and spawning). Three hemocyte types were identified as hyalinocytes and blast-like cells for agranular hemocytes and one granulocyte population. Flow cytometry analysis of hemocytes functionalities indicated that blast-like cells had low oxidative and mitochondrial activities and low lysosomal content. Hyalinocytes and granulocytes are fully equipped to perform innate immune response. Hyalinocytes exhibit higher oxidative activity than granulocytes. Such observation is not common since numerous studies show that granulocytes are usually cells that have the highest cellular activities. This result demonstrates the significant functional variability of hemocyte subpopulations. Moreover, our findings reveal that spawning period of Dreissena polymorpha was associated with an increase of hyalinocyte percentage in relation to low levels of biological activities in hemocytes. This reduction in hemocyte activity would reflect the important physiological changes associated with the spawning period of this invasive species known for its high reproductive potential.

Spatial and temporal variations of biological responses to environmental pollution in the freshwater zebra mussel.

The validation of a suite of cellular biomarkers for biomonitoring studies necessitates a good knowledge of the meaning of these early responses to environmental stress in terms of individual health. This requires confirmation (i) of linkages between the cellular and higher levels of the biological organisation, (ii) of temporal persistence of the stress symptoms and (iii) of their reversibility after a return to more favourable conditions. Besides, (iv) the sensitivity of the biomarker suite towards subtle variations of environmental contamination has to be assessed. With this aim, field experiments were performed on deployed freshwater zebra mussels (Dreissena polymorpha) in the vicinity of the confluence of a small heavily anthropized stream with a larger river. We examined the persistence of the responses over a 90-day period and their reversibility after a depuration-transplantation. A second experiment was conducted later by adding a study site at an increased distance from the confluence. Decreased digestive lysosomal volume and neutral lipid contents, and lipofuscin accumulation preceded effects on the mussels' condition. The following experiment confirmed that the cellular biomarkers were more sensitive than both individual endpoints to reflect the effects of subtler variations of environmental contamination. Integration of the results with multivariate analysis and the Integrated Biomarker Response tended to confirm the relevance of the biomarker suite.

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.

Sperm incorporation and pronuclear development during fertilization in the freshwater bivalve Dreissena polymorpha.

The invasive zebra mussel, Dreissena polymorpha (D. polymorpha), is proving to be a valuable model for understanding general mechanisms of fertilization, particularly regarding sperm incorporation. In the present study, we tracked the various components of the fertilizing sperm of D. polymorpha during sperm incorporation. During fertilization the sperm membrane remains associated with the egg surface as a distinct patch that disperses over time. This patch marked the site of sperm entry that occurs predominately on the CD blastomere. Taking advantage of the relatively unpigmented cytoplasm, real-time observations were made of the incorporated sperm nucleus as it decondensed and reformed as a developing pronucleus. Pronuclear enlargement occurred progressively and at rates comparable with previously reported fixed-time point observations. Sperm mitochondria were incorporated and separated from the sperm along the leading edge of the decondensing nucleus. Sperm mitochondria labeled with Mitotracker Green remained predominately associated with the CD blastomere following first cleavage and could be tracked to the 16-cell stage before the fluorescence was too faint to detect. Additionally, the demembranated sperm axoneme was incorporated, separated during nuclear decondensation, and remained visible in the egg cytoplasm up to 30 min postinsemination (PI). The present study provides one of the most complete descriptions of incorporation on multiple sperm components into the egg and coordinates fixed-time point observations with real-time observations of sperm within the remarkably transparent egg cytoplasm of zebra mussels.

Factors influencing the attachment strength of Dreissena polymorpha (Bivalvia).

The effects of several factors (shell length, exposure time, substratum orientation in space, illumination, temperature, conspecifics) upon the attachment strength (measured with a digital dynamometer) of the freshwater, gregarious bivalve Dreissena polymorpha were studied under laboratory conditions. A rapid increase in attachment strength was observed on resocart (a thermosetting polymer based on phenol-formaldehyde resin, with paper as filler) substrata during the first 4-d exposure, after which it stabilised at ca 1 N. The attachment strength increased also with mussel size. Mussel adhesion on variously oriented surfaces (vertical, upper horizontal and lower horizontal) was similar. Illumination inhibited attachment strength, as expected for a photophobic species, but only after a 2-d exposure. After 6 d, no effects of light were detected. Thus, illumination seemed to influence the attachment rate, rather than the final strength. The optimum temperature for mussel attachment was 20- 25 degrees C. At lower and higher temperatures (5-15 degrees C and 30 degrees C), their adhesion strength decreased. The presence of conspecifics stimulated mussel attachment strength.