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The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus, from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii, and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus, with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host-pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5β, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5β has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5β is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials., (© 2022. The Author(s).)

The present study has investigated the distribution of microplastics in sediment and its impact on histological, ultrastructural, and oxidative stress mechanisms in Perna viridis (P. viridis) from Kasimedu, Chennai, India. The results confirmed that fibers were the predominant type of microplastics observed, followed by spheres, flakes, sheets, and fragments. The observed microplastics were confirmed as polyester, polypropylene, polyethylene, cellophane, and rayon using μ-FT-IR. Microplastic particles entangled in gills caused abrasion of ciliated structure and hemocyte infiltration in the hemolymph vessels. The digestive gland showed a shrunken nucleus, dark inclusions, and damage in the nucleoid core structure. Enlarged vacuoles and the presence of clusters of vesicles presumably represented the transformed golgi cisternae. Further, the results confirmed that oxidative stress markers were significantly high in gills and digestive diverticula of P. viridis. Overall, the results indicated that microplastics induced different toxic physiological and structural alterations in gills and digestive diverticula of P. viridis. These findings highlighted the necessity to focus on exposure studies to understand the absolute magnitude of the problem due to microplastic pollution in the urban estuarine ecosystems of Chennai, Tamil Nadu, India., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis , and the addition of curcumin effectively alleviated the damage induced by P. lima . In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish.

Diarrhetic shellfish toxins (DSTs), some of the most important phycotoxins, are distributed almost all over the world, posing a great threat to human health through the food chain. Therefore, it is of great significance to find effective methods to reduce toxin accumulation in shellfish. In this paper, we observed the effects of four phytochemicals including cinnamaldehyde (CA), quercetin, oridonin and allicin on the accumulation of DSTs in the digestive gland of Perna viridis after exposure to the DSTs-producing Prorocentrum lima . We found that, among the four phytochemicals, CA could effectively decrease the accumulation of DSTs (okadaic acid-eq) in the digestive gland of P. viridis . Further evidence demonstrated that CA could reduce the histological alterations of the digestive gland of a mussel caused by DSTs. RT-qPCR showed that CA could suppress the CYP3A4 induction by DSTs, suggesting that the DSTs' decrease induced by CA might be related to the inhibition of CYP3A4 transcription induction. However, further studies on the underlying mechanism, optimal treatment time, ecological safety and cost should be addressed before cinnamaldehyde is used to decrease the accumulation of DSTs in field.

Microplastics (MPs) are of increasing concern for filter feeding marine and freshwater species. Additionally MPs can sorb hydrophobic contaminants from the water, potentially providing an additional pathway of exposure of aquatic species to contaminants. An acute 48 h laboratory study was conducted to investigate the effects of microplastics and triclosan, both individually and combined, on New Zealand's green-lipped mussel, Perna canaliculus. Biomarkers included clearance rate, oxygen uptake, byssus production; and superoxide dismutase (SOD) activity, glutathione-S-transferase (GST) activity and lipid peroxidation in the gill tissue. Microplastics and triclosan, both individually and combined significantly decreased oxygen uptake and byssus production. These physiological responses were not observed when the microplastics were spiked with triclosan. Triclosan, both alone and spiked to microplastics, increased mussel oxidative stress markers including SOD activity and lipid peroxidation. An enhanced effect was observed on the SOD enzyme activity when mussels were exposed to microplastics spiked with triclosan. No effects on the biochemical biomarkers were observed for mussels exposed to microplastic only. Microplastics enhanced the uptake of triclosan in mussel tissue compared with triclosan only treatments indicating that microplastics potentially provide an additional pathway of exposure to hydrophobic contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

Gills are considered a key player in organism defenses against environmental pollutants. Since it is the major site of uptake of waterborne chemicals, the modulation of important cellular defenses is expected in this tissue. Chlorothalonil, a fungicide presented in herbicides and antifouling paints, might be responsible for toxicity in marine biota. In this context, mussels were exposed to 0.1 μgL -1 and 10 μgL -1 of chlorothalonil for 24 h and 96 h. Genes from biotransformation and antioxidant defense pathways were investigated. Overall, we report, for the first time, an increase in the transcripts of the AhR-like, SULT1A1-like, CYP1A2-like, GSTO-like, MGST-like and SOD-like genes in the gills of the brown mussel Perna perna. This up-regulation was observed mostly after 96 h of exposure to chlorothalonil. Those results reinforce the important role of gills in xenobiotic metabolism and suggest the involvement of the mentioned genes in the detoxification of the compound. Throughout biotransformation and antioxidant defenses pathway, mussels exposed to chlorothalonil are activating mechanisms of defense against this contaminant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Diarrheic shellfish poisoning (DSP) toxins are produced by harmful microalgae and accumulate in bivalve mollusks, causing various toxicity. These toxic effects appear to abate with increasing DSP concentration and longer exposure time, however, the underlying mechanisms remain unclear. To explore the underlying molecular mechanisms, de novo transcriptome analysis of the digestive gland of Perna viridis was performed after Prorocentrum lima exposure. RNA-seq analysis showed that 1886 and 237 genes were up- and down-regulated, respectively after 6 h exposure to P. lima, while 265 genes were up-regulated and 217 genes were down-regulated after 96 h compared to the control. These differentially expressed genes mainly involved in Nrf2 signing pathways, immune stress, apoptosis and cytoskeleton, etc. Combined with qPCR results, we speculated that the mussel P. viridis might mainly rely on glutathione S-transferase (GST) and ABC transporters to counteract DSP toxins during short-term exposure. However, longer exposure of P. lima could activate the Nrf2 signaling pathway and inhibitors of apoptosis protein (IAP), which in turn reduced the damage of DSP toxins to the mussel. DSP toxins could induce cytoskeleton destabilization and had some negative impact on the immune system of bivalves. Collectively, our findings uncovered the crucial molecular mechanisms and the regulatory metabolic nodes that underpin the defense mechanism of bivalves against DSP toxins and also advanced our current understanding of bivalve defense mechanisms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The trace elemental composition of biogenic calcium carbonate (CaCO 3 ) structures is thought to reflect environmental conditions at their time of formation. As CaCO 3 structures such as shell are deposited incrementally, sequential analysis of these structures allows reconstructions of animal movements. However, variation driven by genetics or ontogeny may interact with the environment to influence CaCO 3 composition. This study examined how genetics, ontogeny, and the environment influence shell composition of the bivalve Perna canaliculus. We cultured genetically distinct families at two sites in situ and in the laboratory. Analyses were performed on shell formed immediately prior to harvest on all animals as well as on shell formed early in life only on animals grown in the laboratory. Discriminant analysis using 8 elements (Co, Ti, Li, Sr, Mn, Ba, Mg, Pb, Ci, Ni) classified 80% of individuals grown in situ to their family and 92% to growth site. Generalised linear models showed genetics influenced all elements, and ontogeny affected seven of eight elements. This demonstrates that although genetics and ontogeny influence shell composition, environmental factors dominate. The location at which shell material formed can be identified if environmental differences exist. Where no environmental differences exist, genetically isolated populations can still be identified.

The Byssus, which is derived from the foot gland of mussels, has been proved to bind heavy metals effectively, but few studies have focused on the molecular mechanisms behind the accumulation of heavy metals by the byssus. In this study, we integrated high-throughput transcriptome and proteome sequencing to construct a comprehensive protein database for the byssus of Chinese green mussel (Perna viridis), aiming at providing novel insights into the molecular mechanisms by which the byssus binds to heavy metals. Illumina transcriptome sequencing generated a total of 55,670,668 reads. After filtration, we obtained 53,047,718 clean reads and subjected them to de novo assembly using Trinity software. Finally, we annotated 73,264 unigenes and predicted a total of 34,298 protein coding sequences. Moreover, byssal samples were analyzed by proteome sequencing, with the translated protein database from the foot transcriptome as the reference for further prediction of byssal proteins. We eventually determined 187 protein sequences in the byssus, of which 181 proteins are reported for the first time. Interestingly, we observed that many of these byssal proteins are rich in histidine or cysteine residues, which may contribute to the byssal accumulation of heavy metals. Finally, we picked one representative protein, Pvfp-5-1, for recombinant protein synthesis and experimental verification of its efficient binding to cadmium (Cd2+) ions., Competing Interests: The authors have declared that no competing interests exist.

Blooms of Aureococcus anophagefferens, referred to as brown tides are responsible for massive mortalities and recruitment failure of some bivalves. However, the molecular mechanisms underlying the toxicity remain elusive despite its biological significance, and the information currently available on the molecular effects is still insufficient. In this study, to evaluate the toxicity and associated mechanism of A. anophagefferens on bivalves, we analyzed the protein expression profiles in digestive glands of the A. anophagefferens-exposed Perna viridis by using iTRAQ. A total of 3138 proteins were identified in the digestive glands of A. anophagefferens-exposed P. viridis based on iTRAQ. Amongst, a repertoire of 236 proteins involved in cell, cell part, catalytic activity, metabolic process, biological regulation, immune system process, and response to stimulus were found to be differentially expressed. Functional analysis of the differentially expressed proteins demonstrated that innate immune system of P. viridis was activated, and some proteins associated with stress response and lipid metabolism were induced after exposure to A. anophagefferens. Additionally, MDA content, SOD activity and GSH-Px activity was increased significantly in the digestive gland of A. anophagefferens-exposed P. viridis. Taken together, our results indicated that the A. anophagefferens could induce oxidative stress, activate complement system and alter fat acid metabolism of P. viridis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Chinese green mussel, Perna viridis, is a marine bivalve with important economic values as well as biomonitoring roles for aquatic pollution. Byssus, secreted by the foot gland, has been proved to bind heavy metals effectively. In this study, using the RNA sequencing technology, we performed comparative transcriptomic analysis on the mussel feet with or without inducing by cadmium (Cd). Our current work is aiming at providing insights into the molecular mechanisms of byssus binding to heavy metal ions. The transcriptome sequencing generated a total of 26.13-Gb raw data. After a careful assembly of clean data, we obtained a primary set of 105,127 unigenes, in which 32,268 unigenes were annotated. Based on the expression profiles, we identified 9,048 differentially expressed genes (DEGs) between Cd treatment (50 or 100 μg/L) at 48 h and the control, suggesting an extensive transcriptome response of the mussels during the Cd stimulation. Moreover, we observed that the expression levels of 54 byssus protein coding genes increased significantly after the 48-h Cd stimulation. In addition, 16 critical byssus protein coding genes were picked for profiling by quantitative real-time PCR (qRT-PCR). Finally, we reached a primary conclusion that high content of tyrosine (Tyr), cysteine (Cys), histidine (His) residues or the special motif plays an important role in the accumulation of heavy metals in byssus. We also proposed an interesting model for the confirmed byssal Cd accumulation, in which biosynthesis of byssus proteins may play simultaneously critical roles since their transcription levels were significantly elevated.

Benzo(a)pyrene (BaP) and dichlorodiphenyltrichloroethane (DDT) are persistent organic pollutants and environmental estrogens (EEs) with known toxicity towards the green mussel, Perna viridis. In this study, the toxic effects of BaP (10 µg/L) and DDT (10 µg/L) and their mixture were assessed in green mussel gills with proteomic and metabolomic approaches. Metabolic responses indicated that BaP mainly caused disturbance in osmotic regulation by significantly decrease in branched chain amino acids, dimethylamine and dimethylglycine in gills of male green mussels after exposure for 7 days. DDT mainly caused disturbance in osmotic regulation and energy metabolism by differential alteration of betaine, dimethylamine, dimethylglycine, amino acids, and succinate in gills of male green mussels. However, the mixture of BaP and DDT didn't show obvious metabolite changes. Proteomic analysis showed different protein expression profiles between different treatment groups, which demonstrated that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced cell apoptosis, disturbance in protein digestion and energy metabolism in gills of green mussels, whereas DDT exposure altered proteins that were associated with oxidative stress, cytoskeleton and cell structure, protein digestion and energy metabolism. However, the mixture of BaP and DDT affected proteins related to the oxidative stress, cytoskeleton and cell structure, protein biosynthesis and modification, energy metabolism, growth and apoptosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the current study, we compared protein profiles in gills of Perna viridis after exposure to Prorocentrumlima, a dinoflagellate producing DSP toxins, and identified the differential abundances of protein spots using 2D-electrophoresis. After exposure to P. lima, the level of okadaic acid (a main component of DSP toxins) in gills of P. viridis significantly increased at 6 h, but mussels were all apparently healthy without death. Among the 28 identified protein spots by MALDI TOF/TOF-MS, 12 proteins were up-regulated and 16 were down-regulated in the P. lima-exposed mussels. These identified proteins were involved in various biological activities, such as metabolism, cytoskeleton, signal transduction, response to oxidative stress and detoxification. Taken together, our results indicated that the presence of P. lima caused DSP toxins accumulation in mussel gill, and might consequently induce cytoskeletonal disorganization,oxidative stress, a dysfunction in metabolism and ubiquitination/proteasome activity.

The present study focused on the determination of the alpha-emitter, (210)Po, in two species of marine mussels (bivalve molluscs) commonly available in the southern coastal region of India. The brown mussel, Perna indica was collected from the west coast and the green mussel, Perna viridis from the east coast. The concentration of (210)Po was related to the allometry (length of shell, wet/dry weight of shell/soft tissue) of the mussels and significant results were found. The study period focused on three seasons namely, pre-monsoon, monsoon and post-monsoon for a 1-year period (2010-2011). The results revealed higher activity levels in smaller-sized mussels compared to larger ones. Marked variation in (210)Po activity concentration was noted in the whole-body soft tissues between seasons and sampling site (p < 0.05). The dose rate assessment for mussels was performed using the ERICA Assessment tool. The chronic exposure to mussels due to (210)Po was found to be lesser than the global benchmark dose rate of 10 μGy h(-1). The effective ingestion dose to adults who intake mussels was estimated to be in the range 5.1-34.9 μSv y(-1). The measurement contributes to the furthering of knowledge of (210)Po, since no data exist in this region., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

For evaluating the brown mussel Perna perna as a sentinel organism regarding environmental concentrations of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs), the present study reports original data on the relationship between the concentrations of these chemicals in bottom surface sediments, suspended solids (SS) and concentrations bioaccumulated by this bivalve. Three P. perna cultivation areas, located at three bays in southeastern Brazil were used in this study. The three estuaries are under different degrees of environmental impact. Variations in the OCP and PCB concentrations bioaccumulated by the bivalves tended to be similar to those observed in the sediment, but differed from those found in SS. This latter difference might suggest that the SS trapping apparatuses should have been left in place for approximately 60 days (not only 15 days). This longer period would allow the integration of the environmental variability of the OCP and PCB burden adsorbed to this compartment. Authors encourage future studies to evaluate P. perna exposure to OCPs and PCBs through the evaluation of sediment concentrations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The biochemical responses of the green-lipped mussel, Perna canaliculus, to waterborne cadmium (Cd) were investigated in order to delineate toxic mechanisms, and the impacts of exposure dose and duration, of this important toxicant in a potential sentinel species. Mussels were exposed for either 96 h (acute: 0, 2000, 4000 μgL(-1) Cd) or for 28 d (subchronic: 0, 200, 2000 μgL(-1) Cd), and the digestive gland, gill and haemolymph were examined for impacts. Biochemical responses measured included those associated with metal detoxification (metallothionein-like protein; MTLP), oxidative stress (catalase, lipid peroxidation), cellular homeostasis (alkaline phosphatase, Na(+), K(+)-ATPase; NKA), and energy utilisation (glycogen, haemolymph protein). Following acute exposure, digestive gland glycogen and gill NKA activity were significantly altered by Cd exposure relative to levels in mussels exposed to Cd-free seawater. Subchronic Cd exposure resulted in a significant increase in MTLP levels in both the gill and the digestive gland. This increase was correlated strongly with the levels of Cd accumulation measured in these tissues (R=0.957 for gill, 0.964 for digestive gland). Catalase activity followed a similar pattern, although the correlation with tissue Cd accumulation was not as strong (R=0.907 for gill, 0.708 for digestive gland) as that for MTLP. Lipid peroxidation increased in the digestive gland at Days 7 and 14 at both subchronic Cd levels tested, but this effect had largely dissipated by Days 21 and 28 (with the exception of the 2,000 μgL(-1) group at Day 28). Alkaline phosphatase activity decreased significantly with Cd exposure in both tissues. This effect was observed at both tested concentrations in the gill, but only at the highest concentration for digestive gland. A decrease in digestive gland glycogen levels was observed in Cd-exposed mussels (Days 14 and 21 at 2,000 μgL(-1)), while haemolymph protein levels increased as a result of subchronic Cd exposure. These findings indicated that biochemical responses in Cd-exposed mussels were tissue-specific, dose- and time-dependent, with duration of exposure being the predominant effect. This study shows that biochemical changes in Cd-exposed green-lipped mussels can be linked to tissue metal accumulation and are consistent with previously reported physiological effects. It also suggests that green-lipped mussels are amenable to a multiple biomarker approach and may be of use as a bioindicator species for monitoring coastal metal pollution., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, the aqueous uptake and dietary assimilation (trophic transfer) of two endocrine disrupting compounds (dioxin and phathalic acid) in the green mussel Perna viridis were quantified. During short-term exposure period, dioxin rapidly sorbed onto phytoplankton and its accumulation was much higher than that of phthalate. The uptake of these two compounds by the mussels increased with increasing temperature and salinity (for dioxin only). The dietary assimilation of the two contaminants was rather modest (10-64% for dioxin and 20-47% for phthalate), and was greatly dependent on the food species and concentration. Interestingly, dietary assimilation increased with increasing diatom food concentration. Gut passage time was partially responsible for the variable dietary assimilation. Given the high dissolved uptake rate and the modest dietary assimilation, aqueous exposure was predicted to be the dominant bioaccumulation source for both dioxin and phthalate in the green mussels under most conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although the herbicide glyphosate [N-(phosphonomethyl)glycine] is not classified as an acethylcholinesterase inhibitor, some studies have reported reduction in the acethylcolinesterase activity after in vivo exposure to both its pure form and its commercial formulations. Considering this controversy, the objective of the present study was to investigate, in vitro, the effects of glyphosate exposure on cholinesterase activity of the brown mussel Perna perna and of two fish species: zebrafish Danio rerio and onesided livebearer Jenynsia multidentata. For this purpose, samples of different tissues (brain and muscle for fish; gills and muscle for mussel) were homogenized and pre-incubated with different glyphosate concentrations before cholinesterase activity determination. Results demonstrated that cholinesterase from different fractions of all species tested was inhibited by glyphosate. The concentrations of glyphosate that inhibits 50% of cholinesterase activity (IC50) ranged from 0.62 mM for P. perna muscle to 8.43 mM for J. multidentata brain. According to this, cholinesterase from mussel seems to be more sensitive to glyphosate exposure than those from the fish D. rerio and J. multidentata., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Distribution of heavy metals and its associated histological perturbations were studied in the soft tissues of Perna viridis collected from Ennore estuary and compared with the less polluted Kovalam coast. The concentration of copper, lead, zinc, cadmium, manganese and iron were quantified in gills, digestive gland and adductor muscle. The results showed marked differences between the two sites as well as significant variations within the tissues. Among the heavy metals analyzed, lead and cadmium recorded very low in the soft tissues of mussel. Heavy metal levels in tissues of mussel collected from Ennore estuary were in the order of gills>digestive gland>adductor muscle, while it was digestive gland>gills>adductor muscle in the mussel sampled from Kovalam coast. The decreasing trend of metals in the tissues of mussels sampled from both Ennore estuary and Kovalam coast was in the order of Fe>Mn>Zn>Cu>Pb>Cd. Overall, the highest metal concentrations were found in the mussel collected from Ennore estuary. The metal accumulation in the gills and digestive gland of Perna viridis was found to be quite high in comparison with the adductor muscle. These soft tissues were further investigated by light microscopy and the results were compared with the reference site (Kovalam coast). These results suggest that thickening of the digestive epithelium, hemocytic infiltration in the gills and myodegeneration in the muscle tissue are useful histological biomarkers for heavy metal induced stress, and demonstrate that precautions need to be taken in Ennore estuary in order to prevent heavy metal pollution that can occur in the future., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This study aimed to reveal the proteomic responses in the hepatopancreas and adductor muscle of a common biomonitor, Perna viridis after 14-day exposure to two model chemicals, cadmium (Cd; a toxic metal) and hydrogen peroxide (H(2)O(2); a pro-oxidant), using two-dimensional gel electrophoresis coupled with multivariate statistical analyses. Unique sets of tissue-specific protein expression signatures were revealed corresponding to the two treatment groups. In the hepatopancreas, 15 and 2 spots responded to Cd and H(2)O(2) treatments respectively. 6 and 7 spots were differentially expressed in the adductor muscle for Cd and H(2)O(2) treatments, respectively. 15 differentially expressed spots were successfully identified by MALDI-TOF/TOF MS analysis. These proteins are involved in glycolysis, amino acid metabolism, energy homeostasis, oxidative stress response, redox homeostasis and protein folding, heat-shock response, and muscle contraction modulation. This is the first time, to have demonstrated that Cd exposure not only leads to substantial oxidative stress but also results in endoplasmic reticulum stress in hepatopancreas of the mussel. Such notable stress responses may be attributable to high Cd accumulation in this tissue. Our results suggested that investigations on these stress-associated protein changes could be used as a new and complementary approach in pollution monitoring by this popular biomonitor species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Factorial design plan was experimented in Perna perna in order to find out the contribution of day exposure, Cd and Cu concentrations in water, and their interactions on metallothioneins (MT), Cd, and Cu (the dependent measured variables) in the gills. The picture obtained is more adequate than by studying the factor effect separately. Compared with the control group, the MT concentration after 22 days exposure period in the mixture of 100 μg/mL Cu and Cd is increased almost two times, showing that P. perna might be used as a biomonitor. Cd showed stronger effect than Cu on MT inducing.

The purpose of this study is to conduct a long-term site-specific risk assessment for zinc (Zn) susceptibility of bivalves, green mussel Perna viridis and hard clam Ruditapes philippinarum, based on published experimental data by linking the biologically-based damage assessment model with the subcellular partitioning concept. A comprehensive risk modeling framework was developed to predict susceptibility probability of two bivalve species exposed to waterborne Zn. The results indicated that P. viridis accumulates more Zn toxicity, whereas both toxic potency and the recovery rate of Zn are higher for R. philippinarum. We found that negative linear correlations exist in elimination-recovery and elimination-detoxification relationships, whereas a positive linear correlation was observed in recovery-detoxification relationships for bivalves exposed to waterborne Zn. Simulation results showed that the spatial differences of susceptibility primarily resulted from the variation of waterborne Zn concentration under field conditions. We found that R. philippinarum is more susceptible of Zn than P. viridis under the same exposure condition. Results also suggested that Zn posed no significant susceptibility risk to two bivalve species in Taiwan. We suggested that these two species can be used to biomonitor the water quality on Taiwan coastal areas., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Traditional toxicology studies have focused on selected biomarkers to characterize the biological stress induced by metals in marine organisms. In this study, a system biology tool, metabolomics, was applied to the marine mussel Perna viridis to investigate changes in the metabolic profiles of soft tissue as a response to copper (Cu) and cadmium (Cd), both as single metal and as a mixture. The major metabolite changes corresponding to metal exposure are related to amino acids, osmolytes, and energy metabolites. Following metal exposure for 1 week, there was a significant increase in the levels of branched chain amino acids, histidine, glutamate, glutamine, hypotaurine, dimethylglycine, arginine and ATP/ADP. For the Cu+Cd co-exposed mussels, the levels of lactate, branched chain amino acid, succinate, and NAD increased, whereas the levels of glucose, glycogen, and ATP/ADP decreased, indicating a different metabolic profile for the single metal exposure groups. After 2 weeks of exposure, the mussels showed acclimatization to Cd exposure based on the recovery of some metabolites. However, the metabolic profile induced by the metal mixture was very similar to that from Cu exposure, suggesting that Cu dominantly induced the metabolic disturbances. Both Cu and Cd may lead to neurotoxicity, disturbances in energy metabolism, and osmoregulation changes. These results demonstrate the high applicability and reliability of NMR-based metabolomics in interpreting the toxicological mechanisms of metals using global metabolic biomarkers., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW(1)TAW(2)K and APPPAW(1)TAW(2)K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C(2)-hexosylated tryptophan (W(1)) and C(2)-hexosylated hydroxytryptophan (W(2)), the latter of which is redox active. The UV absorbance spectrum of W(2) is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion.

The biokinetics of p,p'-dichlorodiphenyltrichloroethane (DDT) and its metabolites, p,p'-dichlorodiphenydichloroethylene (DDE) and p,p'-dichlorodiphenyldichloroethane (DDD), in the green-lipped mussel Perna viridis were characterized in this study. We exposed the mussels to DDT in aqueous or dietary sources and then compared and evaluated the absorption, accumulation, distribution and elimination of DDT and its metabolites (DDD and DDE) in the mussels. In addition, a dynamic model was employed to quantify the depuration kinetics of each DDT compound in various organs of the mussels. The potential biotransformation pathway in the mussels after dietary exposure to DDT was also analyzed. Differing accumulation and elimination patterns of each DDT compound (DDT, DDD and DDE) in various organs were observed. Most of the DDT was confined to the hepatopancreas following either aqueous or dietary exposure, although the biological fate and biokinetics of DDT were differed significantly between routes of exposure. In addition, the elimination of dietary DDT was markedly slower than that following aqueous uptake. The biotransformation of DDT to DDE was rare in the mussels, suggesting that any DDE in the mussels came primarily from the ambient environment instead of through biotransformation process. Nevertheless, DDE may be retained in the mussels because of its exceptionally low elimination rate. In contrast, DDT was biotransformed to DDD in the mussels following dietary uptake, and this biotransformation may facilitate DDT elimination from the mussels.

Sub-lethal effects of mercury exposure (110th of LC(50), i.e. 0.045 mg l(-1)) for 5, 10 and 15 d was investigated on oxidative stress parameters and antioxidant defences in digestive gland of Perna viridis. In addition to this an in vitro effect of mercury single and supplemented with reduced glutathione on lipid peroxidation was studied. Increased lipid peroxidation (during first 10 days and also during in vitro exposures), protein carbonyl and hydrogen peroxides (from 5th till last day of exposure) indicate the resultant oxidative stress in the mercury exposed specimen. DNA damage (F-value) response although less distinct on 5th and 15th d, its low values on 10th d and significant correlation with hydrogen peroxide suggests the toxic role of free radicals towards DNA integrity. Superoxide dismutase, which remains low initially (5th d) and increases later suggests its immediate response against superoxide radical. Higher activities of catalase, glutathione peroxidase and glutathione reductase on 15th d and glutathione-S-tranferase from 10th d onwards suggests the adaptive behaviour of the tissue against oxyradicals. Increasing levels of non-enzymatic antioxidant molecules, such as reduced glutathione and ascorbic acid indicated its involvement in counteracting oxidative damage. Further role of reduced glutathione in reducing Hg toxicity is evident in in vitro experiments where lipid peroxidation remains low in mercury concentrations supplemented with reduced glutathione. The elevated levels of metallothionein from 5th to 10th d suggest involvement of this protein in detoxification of reactive oxygen species and toxic metal. The above results suggest that both enzymatic and non-enzymatic antioxidants play an important role in protecting cell against Hg toxicity, which can be used as a biomarker of metal contamination in aquatic environment.

Perna viridis is an ideal animal for studying the impairment caused by the effects of heavy metals that are often detected in coastal areas. Preliminary bioassay tests revealed that the lethal (LC(100)), median lethal (LC(50)) and sublethal (LC(0)) concentration of silver and chromium to P. viridis were 6.5, 4.0, 2.0 mg l(-1) and 4.5, 2.5, 1.0 mg l(-1), respectively. Toxic effect of silver and chromium was evaluated in the green mussel P. viridis, with reference to oxygen consumption, filtration rate and ATPase system in laboratory experiments. These parameters were selected as the end point of sublethal stress. Oxygen consumption and filtration rates were calculated as a measure of decline in the dissolved oxygen level and algal concentration (feed) in the aquaria water, respectively. Silver and chromium affects both oxygen consumption and filtration rate significantly (P<0.01) at 96 h when compared to control. The activity of ATPases system in the gills, hepatopancreas, ovary and muscle of mussels were inhibited by silver and chromium indicating that metals exerted significant toxic effect. The inhibition of Na(+)K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase in the mussels were significant (P<0.05) for silver and highly significant (P<0.01) for chromium, which indicates that chromium was more toxic to mussels when compared to silver. The assessment of oxygen consumption, filtration and ATPases system can thus be used as a valid biomarker in aquatic ecotoxicology studies.

We examined the relationship between Cd kinetics (uptake from solution and diet, and efflux), metallothionein turnover, and changes in the cytosolic distribution of accumulated Cd between protein fractions in the green mussel Perna viridis. We pre-exposed the mussels to 5, 20, 50 and 200 microg l(-1) of Cd for 1 week and determined the biokinetics of Cd uptake and efflux in the mussels. The dietary assimilation efficiency of Cd increased by 2 times following exposure to 20-200 microg l(-1) Cd, but the dissolved uptake rate was unchanged by pre-exposure to any Cd concentrations. The efflux rate of Cd was also similar among control and Cd pre-exposed mussels. The cytosolic distribution of Cd in the mussels that had been exposed to dissolved Cd, showed that besides metallothionein (7000 - 20,000 Da), high molecular weight proteins (>20,000 Da) were important for Cd binding and depuration. In general, the Cd pre-exposed mussels had higher metallothionein turnover with a higher metallothionein synthesis rate, but similar metallothionein breakdown rates as the control mussels. Metallothionein synthesis rate was correlated to the dietary assimilation of Cd, whereas metallothionein breakdown and Cd efflux rate were independent of each other. This study provides important new information for the role of metallothionein turnover on Cd kinetics in an aquatic invertebrate.

Oxidative damage and antioxidant properties have been studied in Perna viridis subjected to short-term exposure to Hg along with temperature (72h) and long-term temperature exposures (14 days) as pollution biomarkers. The elevated thiobarbituric acid reactive substances (TBA-RS) levels observed in gills and digestive gland under exposure to Hg, individually and combined with temperature, as also long-term temperature stress have been assigned to the oxidative damage resulting in lipid peroxidation (LPX). Increased activities of antioxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) both in gills and digestive glands under long-term exposures to temperatures are more prominent to heat rather than cold stress suggesting activation of physiological mechanism to scavenge the ROS produced during heat stress. Also decreased values of reduced glutathione (GSH) on long exposures to temperature stress indicate utilisation of this antioxidant, either to scavenge oxiradicals or act in combination with other enzymes, was more than its production capacity under heat stress. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sublethal effects of pollution.

The presence of pollutants in the ocean may affect different physiological parameters of animals. Oxygen consumption and ammonia excretion were evaluated in D-shaped larvae of mussels (Perna perna) exposed to zinc sulphate (ZnSO(4)) and benzene (C(6)H(6)). When compared to the control group, both pollutants presented a significant reduction in oxygen consumption. A reduction in the ammonia excretion was also observed, both for ZnSO(4) and C(6)H(6) and also in the oxygen consumption. The results indicate that anaerobic metabolism may occur at the beginning of P. perna mussels development, as observed in veliger larvae. The O:N ratio under experimental conditions showed low values indicating that catabolism in veliger larvae was predominantly proteic.

We exposed green-lipped mussels Perna viridis and black sea breams Acanthopagrus schlegeli to toxic dinoflagellates Alexandrium fundyense to evaluate the accumulation, distribution, transformation, and elimination of paralytic shellfish toxins (PSTs) in a controlled environmental condition. The mussels were fed A. fundyense for 7 days followed by 3 weeks of depuration, and the fish were fed toxic clams (pre-exposed to the dinoflagellates) for 5 days followed by 2 weeks of depuration. The toxin content and the compartmental distribution of PSTs were monitored throughout the experiments by high-performance liquid chromatography with post-column fluorescence derivatization (HPLC-FLD). This is the first report to assess the biokinetics of PSTs in marine fish under dietary exposure. The hepatopancreas in the mussels and the viscera in the fish accumulated most of the PSTs. Differential elimination of each toxin was observed in the mussels. The C2 toxins were eliminated rapidly in all organs; except in hepatopancreas, the more potent toxins such as GTX4, were eliminated slower during the depuration period. The relative proportions of various PSTs in the mussels changed over time, suggesting toxin-specific uptake and elimination rates, or biotransformation preferences between toxins. In the fish, the ratio of C1/C2 was 3.0 times (p<0.01) higher when compared to the clam tissues, indicating that conversion from C2 to C1 might have occurred when the toxin was transferred from the clams to the fish. In summary, species differences in uptake, distribution and elimination of PSTs were observed between mussels and fish, and this may influence trophic transfer of algal toxins in marine organisms.

The concentrations of Cu, Pb, and Zn in the crystalline style (CS) and in the remaining soft tissues (ST) of the green-lipped mussel Perna viridis from 10 geographical sites along the coastal waters off peninsular Malaysia were determined. The CS, compared with the remaining ST, accumulated higher levels of Cu in both contaminated and uncontaminated samples, indicating that the style has a higher affinity for the essential Cu to bind with metallothioneins. The similar pattern of Cu accumulation in the different ST of mussels collected from clean and Cu-contaminated sites indicated that the detoxification capacity of the metallothioneins had not been overloaded. For Pb, higher levels of the metal in the CS than in the remaining ST were found only in mussels collected from a contaminated site at Kg. Pasir Puteh. This indicated a tissue redistribution of Pb due to its binding to metallothioneins for Pb detoxification and the potential of the CS as an indicator organ of Pb bioavailability and contamination. For Zn, the above two phenomena were not found since no obvious patterns were observed (lower levels of Zn in the CS than in the remaining ST) in contaminated and uncontaminated samples due to the mechanism of partial regulation. Generally, all the different STs studied (foot, mantle, gonad, CS, gill, muscle, and byssus) are good biomonitoring tissues for Cu and Pb bioavailabilities and contamination. Among these organs, the CS was found to be the best organ for biomonitoring Cu. The present data also suggest the use of the tissue redistribution of Pb in P. viridis as an indicator of Pb bioavailability and contamination in coastal waters.

Physiological studies of the mussel Perna perna in Brazil are almost 30 years behind those of other, more exhaustively investigated species, such as Mytilus edulis. Little is known about the variations in physiological rates due to size and the consequences of maintaining P. perna in laboratory conditions. This work investigated the variations in respiration, clearance, excretion and absorption efficiency rates of P. perna, classified by size and acclimatized in a laboratory, monitoring the mussels respiration rates and biometry over a period of 30 days, in laboratory conditions. The respiration, clearance and excretion rates presented an allometric relation with the dry weight of the organisms, with b values of 0.66, 0.48 and 0.91 respectively. On the other hand, these same rates, when considered by weight (specific rates) showed a relationship that was inverse to the size of the organisms. Only the absorption efficiency was independent of the weight of the mussel. In terms of acclimatization, it was observed that it takes 10 days for the respiration rate of the mussel P. perna to stabilize in laboratory conditions, after which it follows a routine metabolism.

In this study, the green-lipped mussel, Perna viridis (L.), was exposed to two concentrations of benzo[a]pyrene (B[a]P) (0.3 microg l(-1); 3 microg l(-1)) and two concentrations of Aroclor 1254 (0.5 microg l(-1); 5 microg l(-1)). In addition, a mixture of the contaminants was used (0.3 microg l(-1) B[a]P+0.5 microg l(-1) Aroclor 1254; 3 microg l(-1) B[a]P+5 microg l(-1) Aroclor 1254). All concentrations were nominal. A suite of enzymes [glutathione S transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR)], glutathione (GSH) level and lipid peroxidation (LPO) in the mussel gill and hepatopancreas were monitored over 18 days. CAT and GSH in gill tissue were positively correlated with concentration of Aroclor 1254. Activity of hepatic GST and SOD was significantly related to body burden of Aroclor 1254. LPO, GR and GPx in gill and hepatopancreas and hepatic GST were positively correlated with B[a]P concentration. The results indicate the importance of using biomarkers specific to the type of contaminant(s) that are likely to be present. Controlled laboratory experiments, such as this study, are useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment.