Adverse effects of Microcystis aeruginosa exudates on the filtration, digestion, and reproduction organs of benthic bivalve Corbicula fluminea.

Cyanobacteria bloom and the secondary metabolites released by the microorganism are extremely harmful to aquatic animals, yet study on their adverse effects in zoobenthos is rare. Corbicula fluminea widely distributed in freshwater environment with algal blooms. It is a typical filter feeding zoobenthos that may be affected by the secondary metabolites of cyanobacteria due to its high filtering rate. In this study, C. fluminea was exposed to Microcystis aeruginosa exudates (MaE) for 96 h, which was obtained from 5 × 105 cells/mL and 2.5 × 106 cells/mL exponential stage M. aeruginosa culture solution that represented cyanobacteria cell density needs environmental risk precaution control and emergent control, respectively. The responses of C. fluminea critical organs to MaE were analyzed and evaluated based on histopathological sections, antitoxicity biomarkers, and organ function biomarkers. The results showed that all the organs underwent structural disorders, cell vacuolization, apoptosis, and necrosis, and the damage levels increased as MaE concentration increased. The detoxification and antioxidant defense systems biomarkers in each organ response to MaE exposure differently and the level of reaction improved when MaE concentration increased. The siphon rate and acetylcholinesterase activity showed that the filtration function decreased significantly as the MaE concentration increased. Increased activity of glutathione S-transferase and amylase in the digestive gland indicate that it is the major detoxification organ of C. fluminea. Increased vitellogenin concentration and enlarged oocytes in the gonad indicate that MaE may have an estrogenic effect on C. fluminea. This study demonstrates that cyanobacteria threat benthic bivalves by inducing oxidative stress, inhibiting filtering feeding system, and disturbing digestion system and reproduction potential of C. fluminea.

Feeding behavior, microcystin accumulation, biochemical response, and ultramicrostructure changes in edible freshwater bivalve Corbicula fluminea exposed to Microcystis aeruginosa.

As filter-feeders, bivalves naturally come into direct contact with microcystins (MCs) in eutrophic water bodies suffering from cyanobacteria blooms. To date, however, no studies have quantified the dynamics of microcystin accumulation and depuration in the edible freshwater bivalve Corbicula fluminea when exposed to dense bloom concentrations of Microcystis aeruginosa, while considering dynamic changes of biochemical indexes and feeding structure. In the present study, the bioaccumulation and detoxification of microcystin-LR (MC-LR) in C. fluminea were investigated. Our results showed that C. fluminea would graze equally efficiently on green algae and M. aeruginosa, irrespective of whether the M. aeruginosa strains were toxic or non-toxic. MCs could be accumulated and depurated by C. fluminea efficiently. In addition, linear and exposure time-dependent MC-LR accumulation patterns were observed in C. fluminea. Activities of biotransformation (glutathione S-transferase, GST) and antioxidant enzymes (superoxide dismutase, SOD, and catalase, CAT) and malondialdehyde (MDA) contents in various tissues of treated clams were stimulated by MCs in a tissue-specific manner. Our findings indicated that C. fluminea hepatopancreas was the primary target organ for MC-LR detoxification processes, as evidenced by a significant increase in GST activity. Besides, gills and mantle were more sensitive than the other tissues to oxidative stress in the initial microcystin exposure period with a significant increase in SOD activity. The scanning electron microscopy (SEM) observations revealed that the lateral cilia in the gill aperture were well developed during the MCs exposure period, which could perform the filter-feeding function instead of the damaged frontal cilium. This study provides insight into the possible tolerance of C. fluminea exposed to dense bloom concentrations of M. aeruginosa.

Antibacterial properties and cytocompatibility of biobased nanofibers of fish scale gelatine, modified polylactide, and freshwater clam shell.

We report herein new nanofibers prepared from fish scale gelatine (FSG), modified polylactide (MPLA), and a natural antibacterial agent of freshwater clam (Corbicula fluminea Estefanía) shell powder (FCSP). A preparation of FSG from Mullet scales is also described. To improve the biocompatibility and antibacterial activity of the non-woven nanofibers, MPLA/FCSP was added to enhance their antibacterial properties. FSG was then combined with MPLA/FCSP using an electrospinning technique to improve the biocompatibility of the as-fabricated 100-500-nm-diameter non-woven MPLA/FCSP/FSG nanofibers. The resulting tensile properties and morphological characteristics indicated enhanced adhesion among FSG, FCSP, and MPLA in the MPLA/FCSP/FSG nanofibers, as well as improved water resistance and tensile strength, compared with the PLA/FSG nanofibers. MTT assay, cell-cycle, and apoptosis analyses showed that both PLA/FSG and MPLA/FCSP/FSG nanofibers had good biocompatibility. Increasing the FSG content in PLA/FSG and MPLA/FCSP/FSG nanofibers enhanced cell proliferation and free-radical scavenging ability, but did not affect cell viability. Quantitative analysis of bacteria inhibition revealed that FCSP imparts antibacterial activity.

Biofiltration using C. fluminea for E.coli removal from water: Comparison with ozonation and photocatalytic oxidation.

Corbicula fluminea, an Asian clam, is one of the worst invasive species in Europe that can survive in very adverse environmental conditions. Despite its negative impacts, the species also has the capacity to bioaccumulate heavy metals, contaminants and can be exploited for wastewater treatment purposes. The capacity of the Asian clam to remove Escherichia coli, used as fecal contamination indicator, was analyzed. Conventional wastewater treatment plants are not suitable to remove bacteria, thus resulting in treated municipal wastewater with high bacterial loads. E. coli clearance rate was analyzed as function of the number of clams. The bivalves can remove bacteria until concentrations below the detection limit in about 6 h. The adsorption on the clam shells' and bioaccumulation on the soft tissues were also analyzed. The depuration of clams along 48 h were analyzed revealing that no bacteria was detected in the water. Thus, these results suggest that Asian clam can bioprocess E. coli. On the other hand, results obtained by this methodology were compared with ozonation and photocatalytic oxidation using TiO2, Ag, Au, Pd-TiO2. In all treatments it was possible to achieve concentrations of E. coli below the detection limit. However, photocatalytic oxidation demands about 4700 folds more energy than ozonation, besides the costs associated with catalysts. Comparing complexity of ozonation with biofiltration, this study suggests that application of biofiltration using C. fluminea can be a suitable solution to minimize the presence of bacteria in wastewater, reducing environmental and economic impacts.

Effects of benthic macrofauna bioturbation on the bacterial community composition in lake sediments.

Benthic macrofauna are considered to be an important part of the lacustrine ecosystem, and bioturbation may greatly affect the biogeochemical processes and microbial activities in sediments. In the present study, the bacterial community composition in sediments inhabited by 3 different types of benthic macrofauna (Corbicula fluminea, Chironomidae larvae, and tubificid worms) in the shallow and eutrophic Lake Taihu was studied to investigate the different effects of bioturbation on the composition of these communities. Microcosms were constructed, and culture-independent methods, including terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis, were performed to evaluate the bacterial communities. Analysis of similarities (ANOSIM) and multidimensional scaling (MDS) analysis of T-RFLP patterns demonstrated that differences in the bacterial community composition between the control and the macrofauna-inhabited sediments were not as great as expected, although the chemical properties of the sediments changed remarkably. Nevertheless, the dominant bacterial group in each type of macrofauna-inhabited sediment was different. Acidobacteria, Betaproteobacteria, and Deltaproteobacteria were the dominant bacterial groups in sediments inhabited by C. fluminea, tubificid worms, and Chironomidae larvae, respectively. The data obtained in this study are helpful for understanding the effects of bioturbation in a shallow, eutrophic lake.

Differential protein expression in two bivalve species; Mytilus galloprovincialis and Corbicula fluminea; exposed to Cylindrospermopsis raciborskii cells.

The cyanobacteria Cylindrospermopsis raciborskii is considered a threat to aquatic organisms due to the production of the toxin cylindrospermopsin (CYN). Despite the numerous reports evidencing the toxic effects of C. raciborskii cells and CYN in different species, not much is known regarding the toxicity mechanisms associated with this toxin and the cyanobacteria. In this work, a proteomics approach based in the two-dimensional gel electrophoresis and mass spectrometry was used to study the effects of the exposure of two bivalve species, Mytilus galloprovincialis and Corbicula fluminea, to CYN producing (CYN+) and non-producing (CYN-) C. raciborskii cells. Additionally the activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx) were determined. Alterations in actin and tubulin isoforms were detected in gills of both bivalve species and digestive gland of M. galloprovincialis when exposed to CYN- and CYN+ cells. Moreover, GST and GPx activities changed in gills and digestive tract of bivalves exposed to both C. raciborskii freeze dried cells, in comparison to control animals exposed to the green alga Chlorella vulgaris. These results suggest the induction of physiological stress and tissue injury in bivalves by C. raciborskii. This condition is supported by the changes observed in GPx and GST activities which indicate alterations in the oxidative stress defense mechanisms. The results also evidence the capacity of CYN non-producing C. raciborskii to induce biochemical responses and therefore its toxicity potential to bivalves. The heat shock protein 60 (HSP60), extrapallial (EP) fluid protein and triosephosphate isomerase homologous proteins from gills of M. galloprovincialis were down-regulated specifically with the presence of CYN+ C. raciborskii cells. The presence of CYN may lead to additional toxic effects in M. galloprovincialis. This work demonstrates that proteomics is a powerful approach to characterize the biochemical effects of C. raciborskii and to investigate the physiological condition of the exposed organisms.

Dynamics of protein phosphatase gene expression in Corbicula fluminea exposed to microcystin-LR and to toxic Microcystis aeruginosa cells.

This study investigated the in vivo effects of microcystins on gene expression of several phosphoprotein phosphatases (PPP) in the freshwater clam Corbicula fluminea with two different exposure scenarios. Clams were exposed for 96 h to 5 µg L(-1) of dissolved microcystin-LR and the relative changes of gene expression of three different types of PPP (PPP1, 2 and 4) were analyzed by quantitative real-time PCR. The results showed a significant induction of PPP2 gene expression in the visceral mass. In contrast, the cyanotoxin did not cause any significant changes on PPP1 and PPP4 gene expression. Based on these results, we studied alterations in transcriptional patterns in parallel with enzymatic activity of C. fluminea for PPP2, induced by a Microcystis aeruginosa toxic strain (1 × 10(5) cells cm(-3)) during 96 h. The relative changes of gene expression and enzyme activity in visceral mass were analyzed by quantitative real-time PCR and colorimetric assays respectively. The clams exhibited a significant reduction of PPP2 activity with a concomitant enhancement of gene expression. Considering all the results we can conclude that the exposure to an ecologically relevant concentration of pure or intracellular microcystins (-LR) promoted an in vivo effect on PPP2 gene expression in C. fluminea.

Detection of tdh and trh genes in Vibrio parahaemolyticus isolated from Corbicula moltkiana prime in West Sumatera, Indonesia.

The occurrence of Vibrio parahaemolyticus in raw Corbicula moltkiana Prime from Lake Singkarak and Pasar Raya Padang market and in cooked samples in West Sumatera, Indonesia, was studied. Thirteen raw and seven cooked bivalve samples were positive using CHROMAgar Vibrio medium. All 47 V parahaemolyticus isolates were positive for toxR gene but negative for trh. However, 36% (17/47) of V parahaemolyticus strains were positive for tdh gene. Antibiotic profiling showed that 76% and 38% of isolates from raw and cooked bivalves respectively were resistant to ampicillin. Using RAPD-PCR analysis, most of the strains were clustered according to their source of isolation but some of the strains from raw and cooked samples were clustered together. These results indicate that pathogenic V parahaemolyticus isolates are present in Corbicula moltkiana Prime in West Sumatera, Indonesia, suggesting that V parahaemolyticus may also be present in seafood in other regions of Indonesia.

Bacterial tolerances to metals and antibiotics in metal-contaminated and reference streams.

Anthropogenic-derived sources of selection are typically implicated as mechanisms for maintaining antibiotic resistance in the environment. Here we report an additional mechanism for maintaining antibiotic resistance in the environment through bacterial exposure to metals. Using a culture-independent approach, bacteria sampled along a gradient of metal contamination were more tolerant of antibiotics and metals compared to bacteria from a reference site. This evidence supports the hypothesis that metal contamination directly selects for metal tolerant bacteria while co-selecting for antibiotic tolerant bacteria. Additionally, to assess how antibiotic and metal tolerance may be transported through a stream network, we studied antibiotic and metal tolerance patterns over three months in bacteria collected from multiple stream microhabitats including the water column, biofilm, sediment and Corbicula fluminea (Asiatic clam) digestive tracts. Sediment bacteria were the most tolerant to antibiotics and metals, while bacteria from Corbicula were the least tolerant. Differences between microhabitats may be important for identifying reservoirs of resistance and for predicting how these genes are transferred and transported in metal-contaminated streams. Temporal dynamics were not directly correlated to a suite of physicochemical parameters, suggesting that tolerance patterns within microhabitats are linked to a complex interaction of the physicochemical characteristics of the stream.