Cellular and molecular complementary immune stress markers for the model species Dreissena polymorpha.

This study aimed to combine cellular and molecular analyses for better detail the effects of various stresses on a sentinel species of freshwater invertebrate. For this purpose, the hemocytes of the zebra mussel, Dreissena polymorpha, were exposed to different stresses at two different intensities, high or low: chemical (cadmium and ionomycin), physical (ultraviolet B), or biological ones (Cryptosporidium parvum and Toxoplasma gondii). After exposure, flow cytometry and droplet digital PCR analyses were performed on the same pools of hemocytes. Several responses related to necrosis, apoptosis, phagocytosis, production of nitric oxide and expression level of several genes related to the antioxidant, detoxification and immune systems were evaluated. Results showed that hemocyte integrity was compromised by both chemical and physical stress, and cellular markers of phagocytosis reacted to ionomycin and protozoa. While cadmium induced oxidative stress and necrosis, ionomycin tends to modulate the immune response of hemocytes. Although both biological stresses led to a similar immune response, C. parvum oocysts induced more effects than T. gondii, notably through the expression of effector caspases gene and an increase in hemocyte necrosis. This suggests different management of the two protozoa by the cell. This work provides new knowledge of biomarkers in the zebra mussel, at both cellular and molecular levels, and contributes to elucidate the mechanisms of action of different kinds of stress in this species.

From shotgun to targeted proteomics: rapid Scout-MRM assay development for monitoring potential immunomarkers in Dreissena polymorpha.

A highly multiplexed liquid chromatography mass spectrometry-multiple reaction monitoring (MRM)-based assay has been developed for evaluating 107 candidate immune biomarkers in both hemocytes and plasma of the zebra mussel Dreissena polymorpha. The Scout-MRM strategy was employed for the first time, shortening the implementation of a targeted MRM bottom-up proteomics assay using selected immune protein-related peptides identified by shotgun discovery proteogenomics. This strategy relies on spiking scout peptides during the discovery phase and using them to build and deploy the MRM targeted proteomics method. It proved to be highly relevant, since about 90% of the targeted peptides and proteins were monitored and rapidly measured in both hemocyte and plasma samples. The sample preparation protocol was optimized by evaluating the digestion efficiency of tryptic peptides over time. The accuracy and precision of 50 stable isotope-labeled peptides were evaluated for use as internal standards. Finally, the specificity of the transitions was thoroughly assessed to ensure the reliable measurement of protein biomarkers. Several analytical and biological validation criteria were evaluated across hemocytes and plasma samples exposed ex vivo to biological contaminants, resulting in the validation of two Scout-MRM assays for the relative quantitation of 85 and 89 proteins in hemocytes and plasma, respectively. Graphical abstract.

Zebra or quagga mussel dominance depends on trade-offs between growth and defense-Field support from Onondaga Lake, NY.

Two invasive mussels (zebra mussel, Dreissena polymorpha and quagga mussel D. rostriformis bugensis) have restructured the benthic habitat of many water bodies in both Europe and North America. Quagga mussels dominate in most lakes where they co-occur even though zebra mussels typically invade lakes first. A reversal to zebra mussel over time has rarely been observed. Laboratory experiments have shown that quagga mussels grow faster than zebra mussels when predator kairomones are present and this faster growth is associated with lower investment in anti-predator response in quagga mussels than zebra mussels. This led to the hypothesis that the dominance of quagga mussels is due to faster growth that is not offset by higher vulnerability to predators when predation rates are low, as may be expected in newly colonized lakes. It follows that in lakes with high predation pressure, the anti-predatory investments of zebra mussels should be more advantageous and zebra mussels should be the more abundant of the two species. In Onondaga Lake, NY, a meso-eutrophic lake with annual mussel surveys from 2005 to 2018, quagga mussels increased from less than 6% of the combined mussel biomass in 2007 to 82% in 2009 (from 3 to 69% by number), rates typical of this displacement process elsewhere, but then declined again to 11-20% of the mussel biomass in 2016-2018. Average total mussel biomass also declined from 344-524 g shell-on dry weight (SODW)/m2 in 2009-2011 to 34-73 g SODW/m2 in 2016-2018, mainly due to fewer quagga mussels. This decline in total mussel biomass and a return to zebra mussel as the most abundant species occurred as the round goby (Neogobius melanostomus) increased in abundance. Both the increase to dominance of quagga mussels and the subsequent decline following the increase in this molluscivorous fish are consistent with the differences in the trade-off between investment in growth and investment in defenses of the two species. We predict that similar changes in dreissenid mussel populations will occur in other lakes following round goby invasions, at least on the habitats colonized by both species.

Identification of immune-related proteins of Dreissena polymorpha hemocytes and plasma involved in host-microbe interactions by differential proteomics.

Biological responses of zebra mussel Dreissena polymorpha are investigated to assess the impact of contaminants on aquatic organisms and ecosystems. In addition to concentrate chemical contaminants in their tissues, zebra mussels accumulate several microorganisms such as viruses, protozoa and bacteria. In order to understand the molecular mechanisms involved in the defence against microorganisms this study aims at identifying immune proteins from D. polymorpha hemolymph involved in defence against protozoa and viruses. For this purpose, hemolymph were exposed ex vivo to Cryptosporidium parvum and RNA poly I:C. Differential proteomics on both hemocytes and plasma revealed immune proteins modulated under exposures. Different patterns of response were observed after C. parvum and RNA poly I:C exposures. The number of modulated proteins per hemolymphatic compartments suggest that C. parvum is managed in cells while RNA poly I:C is managed in plasma after 4 h exposure. BLAST annotation and GO terms enrichment analysis revealed further characteristics of immune mechanisms. Results showed that many proteins involved in the recognition and destruction of microorganisms were modulated in both exposure conditions, while proteins related to phagocytosis and apoptosis were exclusively modulated by C. parvum. This differential proteomic analysis highlights in zebra mussels modulated proteins involved in the response to microorganisms, which reflect a broad range of immune mechanisms such as recognition, internalization and destruction of microorganisms. This study paves the way for the identification of new markers of immune processes that can be used to assess the impact of both chemical and biological contaminations on the health status of aquatic organisms.

A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes.

Immunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity and phagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations.

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.

First evidence of cytotoxic effects of human protozoan parasites on zebra mussel (Dreissena polymorpha) haemocytes.

The interaction between human protozoan parasites and the immune cells of bivalves, that can accumulate them, is poorly described. The purpose of this study is to consider the mechanisms of action of some of these protozoa on zebra mussel haemocytes, by evaluating their cytotoxic potential. Haemocytes were exposed to Toxoplasma gondii, Giardia duodenalis or Cryptosporidium parvum (oo)cysts. The results showed a cytotoxic potency of the two largest protozoa on haemocytes and suggested the formation of haemocyte aggregates. Thus, this study reveals the first signs of a haemocyte:protozoan interaction.

Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?

The zebra mussel, Dreissena polymorpha constitutes an extensively used sentinel species for biomonitoring in European and North American freshwater systems. However, this invasive species is gradually replaced in freshwater ecosystem by Dreissena bugensis, a closely related dreissenid species that shares common morphological characteristics but possess some physiological differences. However, few are known about differences on more integrated physiological processes that are generally used as biomarkers in biological monitoring studies. Declining of zebra mussel populations raises the question of the sustainability of using one or both species indifferently to maintain the quality of environmental pollution monitoring data. In our study, we performed a field comparative study measuring immune-related markers and bioaccumulation of PCBs, PAHs and PBDEs in sympatrically occurring mussel populations from three sites of the St. Lawrence River. For tested organisms, species were identified using RFLP analysis. Measurement of bioaccumulated organic compounds indicated a higher accumulation of PCBs and PBDEs in D. bugensis soft tissues compared to D. polymorpha while no differences were noticed for PAHs. Results of hemocytic parameters highlighted that differences of hemocyte distributions were associated to modulations of phagocytic activities. Moreover, marked differences occurred in measurement of hemocytic oxidative activity, indicating divergences between the two species for ROS regulation strategies. This physiological characteristic may deeply influence species responses facing environmental or pollution related stress and induce bias if the two species are not differentiated in further biomarker or bioaccumulation measurement-based studies.

Characterization of the multixenobiotic resistance (MXR) mechanism in embryos and larvae of the zebra mussel (Dreissena polymorpha) and studies on its role in tolerance to single and mixture combinations of toxicants.

The study of the cellular mechanisms of tolerance of organisms to pollution is a key issue in aquatic environmental risk assessment. Recent evidence indicates that multixenobiotic resistance (MXR) mechanisms represent a general biological defense of many marine and freshwater organisms against environmental toxicants. In this work, toxicologically relevant xenobiotic efflux transporters were studied in early life stages of zebra mussels (Dreissena polymorpha). Expression of a P-gp1 (ABCB1) transporter gene and its associated efflux activities during development were studied, using qRT-PCR and the fluorescent transporter substrates rhodamine B and calcein-AM combined with specific transporter inhibitors (chemosensitizers). Toxicity bioassays with the model P-gp1 chemotherapeutic drug vinblastine applied singly and in combination with different chemosensitizers were performed to elucidate the tolerance role of the P-gp1 efflux transporter. Results evidenced that the gene expression and associated efflux activities of ABC transporters were low or absent in eggs and increased significantly in 1-3d old trochophora and veliger larvae. Specific inhibitors of Pgp1 and/or MRP transport activities including cyclosporine A, MK571, verapamil and reversin 205 and the musk celestolide resulted in a concentration dependent inhibition of related transport activities in zebra mussel veliger larvae, with IC50 values in the lower micromolar range and similar to those reported for mammals, fish and mussels. Binary mixtures of the tested transporter inhibitors except celestolide with the anticancer drug and P-gp1 substrate vinblastine increased the toxicity of the former compound more than additively. These results indicate that MXR transporter activity is high in early life-stages of the zebra mussel and that may play an important role in the tolerance to environmental contaminants.

Defensin of the zebra mussel (Dreissena polymorpha): molecular structure, in vitro expression, antimicrobial activity, and potential functions.

A 409 bp full length defensin gene was cloned and sequenced based on an expressed sequence tag (EST) obtained from a normalized zebra mussel (Dreissena polymorpha) foot cDNA library developed in our laboratory. The D. polymorpha defensin (Dpd) gene encoded a peptide with 76 amino acid residues. The mature Dpd contains 54 amino acids with a fully functional insect defensin A domain. Homologue searching against GenBank database suggested that this Dpd was phylogenetically close to defensins from a group of insects with six conserved cysteine residues. Predicted with homology-modeling method, the three-dimensional structure of Dpd also demonstrated a significant similarity with insect defensin A. The recombinant Dpd was in vitro expressed through an Escherichia coli expression system. The antimicrobial activities of the refolded recombinant Dpd were found against the growth of Morganella sp., Plesiomonas shigelloides, Edwardsiella tarda, E. coli and Staphylococcus aureus aureus (ATCC12598) with the minimal inhibition concentration (MIC) 0.35, 0.43, 1.16, 6.46, and 30.39 microM, respectively. However, with less than 50 microM no detectable inhibition activities were observed against the other four Gram-negative bacteria (Aeromonas salmonicida salmonicida, Motile Aeromonas, Flavobacterium sp., Pseudomonas fluorescens, Shewanella putrifaciens), as well as the other Gram-positive bacteria (Bacillus megaterium ATCC14581, Carnobacterium maltaromaticum ATCC27865, Enterococcus faecalis ATCC19433, and Micrococcus leteus ATCC4698), and the yeast Saccharomyces cerevisiae. The RNA fluorescent in situ hybridization (FISH) of Dpd in the zebra mussel suggested that the Dpd was expressed in a variety of tissues, such as foot, retractor muscle, ctenidium, mantle, hemocytes, gonad, digestive gland, and intestine. By using the quantitative PCR, the expression level of Dpd in the zebra mussel foot was the highest, followed by the muscle. By comparing the amount of Dpd transcripts in the zebra mussel foot under two different byssogenesis conditions (byssogenesis and non-byssogenesis) with qPCR, we found that the higher expression levels of Dpd were always associated with the byssogenesis status from 2 to 4 days after the start of byssogenesis. Findings of this study suggest that the expression of the Dpd gene was upregulated in vivo by byssogeneis, and by hemocytes in vitro upon stimulation with microbial antigens.

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.

Matrilin-like molecules produced by circulating hemocytes of the zebra mussel (Dreissena polymorpha) upon stimulation.

Since invading the Great Lakes basin, USA, in 1980s, the zebra mussel (Dreissena polymorpha) has caused significant economic and ecologic devastation. The absence of major diseases led to the belief that zebra mussel may have extraordinary host defense mechanisms. This study was undertaken in order to better understand zebra mussel hemocyte functions. A suppressive subtraction hybridization (SSH) cDNA library was constructed from naïve and stimulated hemocytes. Stimulation was performed using a mixture of lipopolysacchride (LPS), peptidoglycan (PGN), and zymosan (ZYM). In the stimulated hemocyte SHH cDNA library, 55 assembled ESTs were differentially expressed. These ESTs contained two putative immune- related molecules, namely, matrilin and agglutinin, three house-keeping genes, six cell metabolism/development genes, and 44 ESTs without putative functions. One of the putative adhesive molecules (CN-29, accession number: AM503947) was predicted to have homology with matrilins including the Von Willebrand Factor A (VWA) domain, which was identical to a matrilin molecule recently reported from the freshwater snail, Biomphalaria glabrata. Preliminary evidence suggests that the zebra mussel matrilin-like molecule is inducible upon hemocyte stimulation.