Copper Stress Causes Shell Morphology Changes in Early Juvenile Anodonta woodiana Based on Geometric-Morphometric Analysis.

In this study, the morphological characteristics of early juvenile shells of Anodonta woodiana, which were exposed to different concentrations of aqueous copper, were analyzed using 10 landmarks to determine morphological changes in the shells. Morphological changes mainly occurred at the top of the shell and front and back ends of the central axis. Stepwise discriminant analysis proved shape differences among experimental and control groups. The results of this study demonstrate for the first time that environmentally relevant copper concentrations cause stress-related morphological changes in A. woodiana in the vulnerable early juvenile stage.

Genotoxic effects of polycyclic aromatic hydrocarbons (PAHs) present in vehicle-wash wastewater on grass carp (Ctenopharyngodon idella) and freshwater mussels (Anodonta cygnea).

Vehicle-wash wastewater (VWW) contains high levels of various petrochemicals such as polycyclic aromatic hydrocarbons (PAHs), a carcinogenic category of organic substances. However, the genotoxic effects of PAHs present in VWW remain largely unknown. We explored the genotoxic effects of PAHs present in VWW on fish grass carp (Ctenopharyngodon idella) and freshwater mussels (Anodonta cygnea). Fish and freshwater mussels were divided into control and exposed groups, the prior groups were treated at weekly intervals with clean water, and the latter with Σ16PAHs contaminated VWW for up to four weeks. The samples of blood from fish and haemolymph from freshwater mussels were collected and analyzed using the comet assay technique. Results exhibited that in control fish and freshwater mussel groups the genotoxicity decreased with every week passing following the order of W1 > W2 > W3 > W4, ranging from 8.33 ± 3.06 to 25.3 ± 4.62 and from 46.0 ± 6.93 to 7.67 ± 3.79, respectively. The exposed fish and freshwater mussel groups indicated an increase in genotoxicity with increasing week intervals with an order of W4 > W3 > W2 > W1, ranging from 55.7 ± 11.9 to 128.3 ± 10.0 and from 112.7 ± 8.50 to 183.3 ± 10.1, respectively. The genotoxic effect of Σ16PAHs on fish was comparatively lower than on freshwater mussels. This study elucidates that VWW is highly genotoxic and should be treated before discharging into aquatic ecosystems.

Anodonta cygnea, a freshwater swan mussel, exposed to diazinon: toxicity thresholds in behaviour and physiology.

Swan mussels (Anodonta cygnea) have been suggested as suitable bioindicators for the presence of pollutants in the environment. Application of the physiological and behavioral markers in these sessile species can be beneficial for environmental monitoring. The present study aimed to investigate the relationship between the behavioral disorders of movement and siphoning associated with the inhibition of tissue Acetylcholinesterase (AChE). For experiments, overally 120 bivalves of Anodonta cygnea (mean total length 80.33 ± 6.7 mm) were transported from the agricultural drains and canals in Sari county (Mazandaran Province, Iran) to our laboratory. First, the LC50-96 h of diazinon was estimated according to the Organization for Economic Co-operation and Development (OECD 1992) guideline with static water conditions. The sub-lethal toxicity pesticide experiments were conducted on the basis of the lowest observed effect concentration (LOEC) and the maximum acceptable toxicant concentration (MATC). The LC50-96 h, LOEC, and MATC values of diazinon were 85.2, 42.1, and 8.5 mg L- 1, respectively. Based on the observations of mussels' movement, the burrowing and displacement decreased with the concentration of toxicant in water. Moreover, the presence of diazinon in water and its exposure to experimental animals significantly reduces their siphoning rate. The RDA showed that the AChE activity had a higher correlation with the siphoning behavior than the movement behavior. The comparison of enzyme activity at different exposure and recovery times showed that there was a significant difference among the groups affected by the consumed pesticide (p = 0.001, between contrasts). The most remarkable morphometric characteristic was the siphon opening that was inversely correlated with the enzymatic activity. Studies in bioethics might benefit from paying attention to these traits that are directly related to the level of toxicity and behavioral adaptations required for animal survival.

Molecular Characterization of MyD88 in Anodonta woodiana and Its Involvement in the Innate Immune Response to Bacterial Infection.

Myeloid differentiation factor 88 (MyD88) is a key adapter molecule in Toll-like receptor signal transduction that triggers downstream immune cascades involved in the host defense response to exogenous pathogens. However, the function of MyD88s in mollusks, especially in freshwater shellfish, remains poorly understood. In this study, a novel freshwater shellfish MyD88 (denoted AwMyD88) was characterized from Anodonta woodiana. The present AwMyD88 protein consists of 474 amino acids and contains a conserved a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain with three typical boxes. Quantitative real-time PCR (qRT-PCR) analysis showed that AwMyD88 was broadly expressed in all the examined tissues, and the highest expression level was observed in hemocytes of A. woodiana. When challenged with Aeromonas hydrophila and lipopolysaccharide (LPS), the mRNA expression levels of AwMyD88 were significantly induced in hemocytes of A. woodiana in vivo and in vitro. In addition, in vivo injection experiments revealed that MyD88 signaling pathway genes showed strong responsiveness to A. hydrophila challenge, and their expression levels were significantly upregulated in hemocytes. Knockdown of AwMyD88 reduced the transcript levels of immune related transcription factors (AwNF-κB and AwAP-1) and effectors (AwTNF, AwLYZ, AwDefense and AwAIF) during A. hydrophila infection. Moreover, subcellular localization analysis indicated that AwMyD88 was mainly localized to the cytoplasm in HEK293T cells. Finally, luciferase reporter assays revealed that AwMyD88 associates with AwTLR to activate the NF-κB and AP-1 signaling pathways in HEK293T cells. These results suggested that AwMyD88 might be involved in the host defense response to bacterial challenge, providing new insight into the immune function of the MyD88 signaling pathway in freshwater shellfish.

The impact of iron coagulant on the behavior and biochemistry of freshwater mussels Anodonta cygnea and Unio tumidus during lake restoration.

Iron (Fe) treatment is one of the most commonly used methods to restore eutrophic lakes and reservoirs. The Fe-based coagulants dosage results in an almost immediate improvement in water quality at a relatively low cost. However, the effects of the application of coagulants are not always predictable, and the scale of the risks is not fully understood. The dosage of coagulants changes the chemical and physical properties of water, thereby affecting aquatic biocenoses. In this study, several laboratory experiments were conducted to evaluate the effects of Fe-based coagulant dosage on two bivalves species: Anodonta anatina and Unio tumidus. Their ability to efficiently filter water and reduce seston makes them a key component of aquatic ecosystems in terms of maintaining proper ecological health and stable functioning. Behavioral response, biochemical parameters, and body chemistry changes in mussels exposed to different doses of coagulant were surveyed. A dose-dependent reduction in filtration activity of both species was observed. As early as 10 g Fe m2 (which is a moderate dose used in lakes restoration), mussels of both species almost completely reduced their filtration activity and remained with closed valves for several subsequent days. Significant Fe accumulation in muscles of bivalves exposed to coagulant was also observed. This was particularly the case when very high doses of coagulant were applied. Then, the iron content in leg muscles of both species increased over fourfold. At the same time, a decrease in muscles calcium and phosphorus content was observed. No symptoms of oxidative stress (TBARS, H2O2) after mussels exposure to coagulants were found. The results suggest that the application of Fe-based coagulant for water ecosystem restoration may be a threat to the mussels population. These findings are significant for decisions on the selection of restoration methods for a specific lake.

Contrasting temperature responses in seasonal timing of cercariae shedding by Rhipidocotyle trematodes.

Global warming is likely to lengthen the seasonal duration of larval release by parasites. We exposed freshwater mussel hosts, Anodonta anatina, from 2 high-latitude populations to high, intermediate and low temperatures throughout the annual cercarial shedding period of the sympatric trematodes Rhipidocotyle fennica and R. campanula, sharing the same transmission pathway. At the individual host level, under warmer conditions, the timing of the cercarial release in both parasite species shifted towards seasonally earlier period while its duration did not change. At the host population level, evidence for the lengthening of larvae shedding period with warming was found for R. fennica. R. campanula started the cercarial release seasonally clearly earlier, and at a lower temperature, than R. fennica. Furthermore, the proportion of mussels shedding cercariae increased, while day-degrees required to start the cercariae shedding decreased in high-temperature treatment in R. fennica. In R. campanula these effects were not found, suggesting that warming can benefit more R. fennica. These results do not completely support the view that climate warming would invariably increase the seasonal duration of larval shedding by parasites, but emphasizes species-specific differences in temperature-dependence and in seasonality of cercarial release.

Identification and characterization of MKK6 and AP-1 in Anodonta woodiana reveal their potential roles in the host defense response against bacterial challenge.

Mitogen-activated protein kinase kinase 6 (MKK6) and activator protein-1 (AP-1) are two of the essential regulatory proteins in the p38 mitogen-activated protein kinase (MAPK) pathway, which participates in the innate immune response to bacterial infections. In this study, molluscan MKK6 (AwMKK6) and AP-1 (AwAP-1) genes were cloned and identified from Anodonta woodiana. The open reading frame (ORF) of AwMKK6 encodes for a putative polypeptide sequence of 345 amino acids containing a conserved serine/threonine protein kinase (S_TKc) domain, a SVAKT motif and a DVD domain. AwAP-1 consists of 294 amino acids including a typical nuclear localization signal (NLS), a Jun domain and a basic region leucine zipper (BRLZ) domain. Quantitative real-time PCR analysis showed that both AwMKK6 and AwAP-1 were widely expressed in all selected tissues of A. woodiana and their transcript levels in hemocytes were significantly upregulated when challenged with Aeromonas hydrophila and lipopolysaccharide (LPS). Additionally, the signaling molecules of the AwMKK6/AwAP-1 pathway including AwTLR4, AwMyD88, AwTRAF6, AwMEKK1, AwMEKK4, AwASK1, AwTAK1 and Awp38 mRNA expression showed a stronger responsiveness to LPS challenge in hemocytes of A. woodiana. RNA interference (RNAi) experiments indicated that the silencing of AwMKK6 or AwAP-1 could decrease the mRNA expression levels of immune effectors (AwTNF, AwLYZ and AwDefense). Subcellular localization studies suggested that AwMKK6 and AwAP-1 were distributed throughout the cells and nucleus, respectively, and their overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. These findings suggest that MKK6 and AP-1 play a major role in the host defense response to bacterial injection, which may make contributions to a better understanding of the immune function of the p38 MAPK pathway in mollusks.

Evaluation of transcriptional biomarkers using a high-resolution regression approach: Concentration-dependence of selected transcripts in copper-exposed freshwater mussels (Anodonta anatina).

We tested concentration-dependence of selected gene transcripts (cat, gst, hsp70, hsp90, mt and sod) for evaluation as biomarkers of chemical stress. Contrary to the common approach of factorial designs and few exposure concentrations, we used regression across a high-resolution concentration series. Specifically, freshwater mussels (Anodonta anatina) were acutely (96 h) exposed to Cu (13 nominal concentrations, measuring 0.13-1 600 µg/L), and transcripts were measured by RT-qPCR. In digestive glands, cat, hsp90 and mt decreased with water Cu (p < 0.05), but response magnitudes saturated at < 2-fold decreases. In gills, gst, hsp70, hsp90 and mt increased with water Cu (p < 0.05). While hsp70, hsp90 and mt exceeded 2-fold increases within the exposure range, high Cu concentrations were required (38-160 µg/L). Although gill responses were generally more robust compared to digestive glands, overall small response magnitudes and moderate sensitivity may set limit for potential application as general biomarkers of chemical stress.

Cadmium-induced ultrastructural changes and apoptosis in the gill of freshwater mussel Anodonta woodiana.

This study investigated the acute toxicity of cadmium (Cd) to the freshwater mussel Anodonta woodiana. The freshwater mussels were exposed to five concentrations of Cd (0 mg/L, 8.43 mg/L, 16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) for up to 96 h. The 24-h, 48-h, 72-h, and 96-h LC50 values for Cd were estimated as 562.3 mg/L, 331.1 mg/L, 182.0 mg/L, and 134.9 mg/L, respectively. Caspase-3, caspase-8, caspase-9, and Ca-ATPase activities; protein and H2O2 levels; DNA fragmentation; and ultrastructure of the gill were also investigated. The activities of caspase-3 and caspase-9 in mussels were increased by Cd in a dose-dependent manner, where higher doses of Cd (33.72 mg/L and 67.45 mg/L) significantly increased the enzyme activities compared to the controls (P < 0.05). The caspase-8 activity was significantly depressed by a low dose of Cd (8.43 mg/L) but was clearly induced by higher doses of Cd (16.86 mg/L, 33.72 mg/L, and 67.45 mg/L) (P < 0.05). The Ca-ATPase activity and H2O2 levels were elevated and reached maximum values under the medium dose of Cd (16.86 mg/L). However, protein levels were decreased by Cd in an inverse dose-dependent manner. In the gills of the mussels, Cd treatment induced DNA fragmentation as demonstrated by DNA ladders observed via agarose gel electrophoresis. Moreover, ultrastructural alterations in gill cells of mussels treated with Cd (16.86 mg/L and 67.45 mg/L) for 96 h were observed by electronic microscopy. The ultrastructure abnormalities were characterized by the following features: (1) a disordered arrangement and breaking off of microvilli of epithelial cells; (2) chromatin condensed near the nuclear membrane and the appearances of extremely irregular nuclei, some with a fingerlike shape and an unclear, swollen, invaginated, or ruptured nuclear membrane and apoptotic bodies; (3) swollen and vacuolating mitochondria, some with disintegrated or missing cristae; (4) a disintegrated rough endoplasmic reticulum containing different sizes of vesicles; and (5) shrinking and deformation of Golgi bodies with decreased vesicle numbers. Our results demonstrated that Cd could activate caspase-3, caspase-8, caspase-9, and Ca-ATPase; cause ultrastructural changes; and produce DNA fragmentation in the mussels investigated. Based on the information obtained through this study, it is reasonable to conclude that Cd can induce apoptosis in the gills of the mussels, eventually leading to tissue damage.

Molecular biomarker responses in the freshwater mussel Anodonta anatina exposed to an industrial wastewater effluent.

Using a selection of molecular biomarkers, we evaluated responses in freshwater mussels (Anodonta anatina) exposed to effluent from an industrial wastewater treatment facility. The aims of this work were to (1) assess biomarkers of general toxicity under sublethal exposure to an anthropogenic mixture of chemicals, represented by an arbitrary effluent, and (2) evaluate the potential of A. anatina as a bioindicator of pollution. Adult mussels (n = in total 32; 24 males and 8 females) were exposed (96 h) in the laboratory to a fixed dilution of effluent or to a control treatment of standardized freshwater. Metal concentrations were in general higher in the effluent, by an order of magnitude or more, compared to the control. Toxic unit estimates were used as proxies of chemical stress, and Cu, Ni, and Zn were identified as potential major contributors (Cu> Ni > Zn). Six transcriptional (cat, gst, hsp70, hsp90, mt, sod) and two biochemical (AChE, GST) biomarkers were analyzed in two tissues, gills, and digestive glands. Out of the 16 responses (eight biomarkers × two tissues), 14 effect sizes were small (within ± 28 % of control) and differences non-significant (p > 0.05). Results did however show that (1) AChE activity increased by 40% in gills of exposed mussels compared to control, (2) hsp90 expression was 100% higher in exposed female gills compared to control, and (3) three marker signals (AChE in both tissues, and hsp70 in gills) differed between sexes, independent of treatment. Results highlight a need for further investigation of molecular biomarker variability and robustness in A. anatina.

Survival and spawning success of American shad (Alosa sapidissima) in varying temperatures and levels of glochidia infection.

Temperature fluctuations and climate change impacts may substantially affect spawning success of fish, especially migratory species with a limited spawning window. Factors affecting American shad (Alosa sapidissima) spawning success and survival were investigated at different temperatures and periods (peak- and late-spawning periods) during the Connecticut River, USA, spawning migration in 2017. Wild caught American shad were exposed to constant temperatures regimes of 15, 18, 21, 24 and 27 °C for 2 weeks. During the peak-spawning period, an increase in temperature (15-24 °C) was shown to increase spawning success factors, including spawning probability, number of eggs, and fertilization success, but decreased egg size. Temperatures between 18 and 27 °C did not affect these factors during the late-spawning period. Glochidia infection by the alewife floater (Anodonta implicata) was much higher in the late-spawning period and significantly decreased the survival of American shad. Further research should investigate the parasite-host relationship between the alewife floater and American shad to determine annual variability of glochidia infections and how they affect American shad from physiological and passage perspectives. Higher temperatures were shown to increase spawning success of American shad during the peak-spawning period, but temperature had no effect during the late-spawning period. However, any effect during the late-spawning period may have been masked by a high level of glochidia infection.

Importance of substratum quality for potential competitive niche overlap between native and invasive unionid mussels in Europe.

Infaunal freshwater mussels are highly threatened and declining worldwide. One of the potential threats to mussels consists of biological invasions. We intended to investigate the habitat overlap and behavioural differences between native (Unio pictorum, Unio tumidus, Anodonta anatina, Anodonta cygnea) and invasive (Asian Sinanodonta woodiana) unionid bivalves to determine potential sources of competition. Furthermore, we investigated differences between S. woodiana from the established population in artificially heated waters and from the recent population in a natural thermal regime. We used pairwise choice tests on mud, medium, coarse and very coarse sand, mixture of medium and coarse sand, fine, medium and coarse gravel, and observed mussel locomotion and burrowing in preferred and non-preferred substrata. All species generally preferred fine-grained materials. The widest preference range was exhibited by S. woodiana (both populations), whereas A. cygnea was the most selective. The preferences of the cold-water population of S. woodiana were shifted towards coarser materials compared to conspecifics from the heated waters, and highly overlapped with the preferences of the native species. Anodonta cygnea most often moved horizontally and spent the shortest time deeply burrowed. Both Unio species were deeply burrowed for the largest amount of time and the horizontal locomotion of U. tumidus was the lowest among the test species. Sinanodonta woodiana, especially from the heated water population, exhibited relatively weak locomotion (compared to A. cygnea) and burrowing (compared to Unio spp. and A. anatina). Deep burrowing was more common on fine-grained materials. Our results suggest that the native mussels can be threatened by S. woodiana due to their overlapping habitat preferences, potentially hindering habitat separation. However, mobile native mussels may be capable of migrating and avoiding competition. Accumulating knowledge of the biology and ecology of freshwater mussels could contribute to the creation and improvement of conservation plans to protect these threatened animals.

The joint adverse effects of aged nanoscale plastic debris and their co-occurring benzo[α]pyrene in freshwater mussel (Anodonta anatina).

Although the presence of small-scale plastics, including nanoscale plastic debris (NPD, size <1 µm), is expected in the environment, our understanding of their potential uptake and biodistribution in organisms is still limited. This mostly is because of the limitations in analytical techniques to characterize NPD in organisms' bodies. Moreover, it is still debatable whether aged NPD can sorb and transfer chemicals into organisms. Here, we apply iron oxide-doped polystyrene nanoparticles (Fe-PS NPs) of 270 nm size to quantify the uptake and biodistribution of NPD in freshwater mussels (Anodonta anatina). The Fe-PS NPs were, first, oxidized using heat-activated potassium persulfate treatments to produce NPD (aged particles). Then, the sorption of benzo[a]pyrene (B[α]P), as a model of organic chemicals, into the aged NPD was studied. Chemical oxidation (i.e. aging) significantly decreased the sorption of B[α]P into the particles over 5 days when compared to pristine particles. After 72-h of exposure, A. anatina accumulated NPD in the gills and digestive gland. When exposed to the mixture of NPD and B[α]P, the number of particles in the gills and digestive gland increased significantly compared to the mussels exposed to NPD alone. Moreover, the mixture of NPD and B[α]P increased the activity of Superoxide dismutase and Catalase enzymes in the exposed mussels when compared to the control and to the NPD alone. The present study provides evidence that aged NPD not only could accumulate and alter the toxicity profile of organic chemicals in aquatic organisms, but the chemicals also could facilitate the uptake of NPD (combined effects).

Chitinibacter bivalviorum sp. nov., isolated from the gut of the freshwater mussel Anodonta arcaeformis.

A novel Gram-stain-negative, aerobic, rod-shaped bacterium with a single polar flagellum, designated strain 2T18T, was isolated from the gut of the freshwater mussel Anodonta arcaeformis collected in the Republic of Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Chitinibacter. Strain 2T18T formed a monophyletic clade with Chitinibacter fontanus KCTC 42982T, C. tainanensis KACC 11706T and C. alvei KCTC 23839T, with sequence similarities of 98.5, 98.4 and 95.9 %, respectively. Strain 2T18T exhibited optimal growth at 30 °C, at pH 8 and with 0.5 % (w/v) NaCl. The major isoprenoid quinone was ubiquinone-8 (Q-8). The predominant fatty acids were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified lipid, three unidentified phospholipids and two unidentified aminophospholipids. The G+C content of the genomic DNA was 50.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strains 2T18T and C. fontanus KCTC 42982T were below the thresholds used for the delineation of a novel species. Based on the phylogenetic, phenotypic, chemotaxonomic and genotypic characteristics, strain 2T18T represents a novel species of the genus Chitinibacter, for which the name Chitinibacter bivalviorum sp. nov. is proposed. The type strain is 2T18T (=KCTC 72821T=CCUG 74764T).

Cadmium bioaccumulation and distribution in the freshwater bivalve Anodonta woodiana exposed to environmentally relevant Cd levels.

Anodonta woodiana is a globally distributed freshwater bivalve, which is a unique bioindicator in the "Freshwater Mussel Watch" project. Numerous countries have used A. woodiana for biomonitoring the aqueous cadmium (Cd) contamination. However, the bioaccumulation and distribution characteristics of environmentally relevant Cd concentrations in the bivalve were unknown. In this study, A. woodiana was exposed to aqueous Cd concentrations (1.1, 2.6, and 5.5 µg/L) for 30 days. The concentrations of Cd in the whole soft tissues were linearly and positively correlated with the aqueous Cd concentrations and exposure time (P < 0.05). Analysis of the organic bioaccumulation and distribution showed that Cd concentrations and proportions in the gills and mantle were linearly and positively correlated with aqueous Cd concentrations and with the Cd concentrations in the whole soft tissues (P < 0.05). Analyses of the subcellular fractions showed that Cd concentrations and proportions in the metal-rich granule (MRG) were linearly and positively correlated with aqueous Cd concentrations and with the Cd concentrations in the whole soft tissues (P < 0.05). This suggests that the gills and mantle could be the main target organs for Cd bioaccumulation, and that the MRG could be the major site for Cd bioaccumulation and distribution. These results improve our understanding of the bioaccumulation and distribution mechanisms of Cd in bivalves.

Modelling the habitat preferences of the swan mussel (Anodonta cygnea) using data-driven model.

The Anzali wetland (located in northern Iran) and many parts of its catchment are considered important habitats for the swan mussel (Anodonta cygnea). The habitat of this native bioindicator mussel is being threatened in many locations of the catchment due to various anthropogenic activities. The present study aimed to apply a classification tree model (J48 algorithm) to predict the habitat preferences of A. cygnea in 12 sampling sites based on various water quality and physical-habitat variables. The species was present in 50% of sampling sites, while it was absent in the remaining of the sampling sites. In total, 144 samples of A. cygnea (72 presence and 72 absence instances) were monthly measured together with the abiotic variables during 1-year study period (2017-2018). For the CT model, two-thirds of datasets (96 instances) served as a training and the remainder was employed for the validation set (48 instances). Among 25 environmental variables introduced to the model (with pruning confidence factor = 0.10, threefold cross-validation and 5 times randomization effort), the validity of 6 variables was confirmed by the model in all three subsets. Water salinity, flow velocity, water depth and water turbidity were jointly predicted by the model in three subsets. The model predicted that the absence of A. cygnea might be associated with increasing flow velocity, total phosphate and water turbidity. In contrast, the presence of A. cygnea might be related to decreased water depth and increased calcium concentration. The model also confirmed that all predicted variables for the species might be completely dependent on the water salinity. According to the chi-square test (x2 = 26.53, p < 0.05), the habitat condition of A. cygnea is influenced by significant variations in the spatio-temporal patterns.

Transcriptional and biochemical biomarker responses in a freshwater mussel (Anodonta anatina) under environmentally relevant Cu exposure.

Molecular biomarkers, like gene transcripts or enzyme activities, are potentially powerful tools for early warning assessment of pollution. However, a thorough understanding of response and baseline variation is required to distinguish actual effects from pollution. Here, we assess the freshwater mussel Anodonta anatina as a biomarker model species for freshwater ecosystems, by testing responses of six transcriptional (cat, gst, hsp70, hsp90, mt, and sod) and two biochemical (AChE and GST) biomarkers to environmentally relevant Cu water concentrations. Mussels (n = 20), collected from a stream free from point source pollution, were exposed in the laboratory, for 96 h, to Cu treatments (< 0.2 µg/L, 0.77 ± 0.87 µg/L, and 6.3 ± 5.4 µg/L). Gills and digestive glands were extracted and analyzed for transcriptional and biochemical responses. Biological and statistical effect sizes from Cu treatments were in general small (mean log2 fold-change ≤ 0.80 and Cohen's f ≤ 0.69, respectively), and no significant treatment effects were observed. In contrast, four out of eight biomarkers (cat, gst, hsp70, and GST) showed a significant sex:tissue interaction, and additionally one (sod) showed significant overall effects from sex. Specifically, three markers in gills (cat, mt, GST) and one in digestive gland (AChE) displayed significant sex differences, independent of treatment. Results suggest that sex or tissue effects might obscure low-magnitude biomarker responses and potential early warnings. Thus, variation in biomarker baselines and response patterns needs to be further addressed for the future use of A. anatina as a biomarker model species.

Usability of the bivalves Dreissena polymorpha and Anodonta anatina for a biosurvey of the neurotoxin BMAA in freshwater ecosystems.

The environmental neurotoxin ß-methylamino-L-alanine (BMAA) may represent a risk for human health in case of chronic exposure or after short-term exposure during embryo development. BMAA accumulates in freshwater and marine organisms consumed by humans. It is produced by marine and freshwater phytoplankton species, but the range of producers remains unknown. Therefore, analysing the phytoplankton composition is not sufficient to inform about the risk of freshwater contamination by BMAA. Filter-feeders mussels have accumulation capacities and therefore appear to be relevant to monitor various pollutants in aquatic ecosystems. We investigated the suitability of the freshwater mussels Dreissena polymorpha and Anodonta anatina for monitoring BMAA in water. Both species were exposed to 1, 10, and 50 µg of dissolved BMAA/L daily for 21 days, followed by 42 days of depuration in clean water. On days 0, 1, 7, 14, and 21 of exposure and 1, 7, 14, 21 and 42 of depuration, whole D. polymorpha and digestive glands of A. anatina were sampled, and the total BMAA concentration was measured. D. polymorpha accumulated BMAA earlier (from day 1 at all concentrations) and at higher tissue concentrations than A. anatina, which accumulated BMAA from day 14 when exposed to 10 µg BMAA/L and from day 7 when exposed to 50 µg BMAA/L. As BMAA accumulation by D. polymorpha was time and concentration-dependent, with a significant elimination during the depuration period, this species may be able to reflect the levels and dynamics of water contamination by dissolved BMAA. The species A. anatina could be used for monitoring water concentrations above 10 µg BMAA/L.

Molecular characterization of AwSox2 from bivalve Anodonta woodiana: Elucidating its player in the immune response.

Sox2 is an embryonal stem cell Ag essential for early embryonic development, tissue homeostasis and immune regulation. In the current study, one complete Sox2 cDNA sequence was cloned from freshwater bivalve Anodonta woodiana and named AwSox2. Histological changes of testis derived from Bisphenol A (BPA) treatment were analyzed by hematoxylin and eosin staining. Expressions of AwSox2 derived from BPA, LPS and polyinosinic:polycytidylic (Poly I:C) challenge were measured by quantitative real-time PCR. The full-length cDNA of AwSox2 contained an open reading frame of 927 nucleotides bearing the typical structural features of Sox2 family. Obvious degeneration, irregular arrangement of spermatids, and clotted dead and intertwined spermatids were observed in BPA-treated groups. Administration of BPA could result in a dose-dependent up-regulation of AwSox2 expression in the male gonadal tissue of A. woodiana. In addition, expression of AwSox2 was significantly induced by LPS and Poly I:C treatment in the hepatopancreas, gill and hemocytes, compared with that of control group. These results indicated that up-regulations of AwSOx2 are closely related to apoptosis of spermatogonial stem cells derived from BPA treatment as well as enhancement of immune defense against LPS and Poly I:C challenge in A. woodiana.

Potential impacts of the invasive species Corbicula fluminea on the survival of glochidia.

Freshwater mussels (Bivalvia, Unionida) are one of the most imperilled faunal groups globally, being the introduction of invasive species a possible major mechanism of threat. The Asian clam Corbicula fluminea is a problematic invasive species in aquatic ecosystems and can impair the survival of parasitic larvae (glochidia) of native freshwater mussels. However, this possible mechanism of threat remains speculative and to date very few studies addressed quantitatively this issue. In order to cover this gap, we have performed a series of manipulative laboratory studies to assess how distinct densities of C. fluminea can affect the survival of glochidia after 6, 12, 24 and 48 h of exposure, using larvae of the native freshwater mussel Anodonta anatina. Our results suggest an increase in mortality of A. anatina glochidia with an increase in density of C. fluminea. Two main mechanisms may possibly explain our results: 1) the high filtration capacity of C. fluminea that can contribute to the mortality of glochidia due to the mechanical damage of their fragile shells when passing by siphons and/or digestive tract of C. fluminea and 2) the high excretion capacity of C. fluminea that can lead to mortality of glochidia due to increase in ammonia concentration. Mortality of glochidia was also time dependent with higher values registered after 48 h. This work is one of the first showing the influence of C. fluminea density on the survival of glochidia, being filtration (and consequent passage in the digestive tract) and biodeposition the main potential mechanisms explaining overall mortality. These results also suggest that sites with high densities of C. fluminea may be highly detrimental for the conservation of freshwater mussels, potentially impairing the survival of glochidia and negatively affecting the recruitment of juveniles.