Physiological and biochemical response in green mussel Perna viridis subjected to continuous chlorination: Perspective on cooling water discharge criteria.

Heavy infestation by Perna viridis has been observed in the sub-seabed seawater intake tunnel and CWS of a tropical coastal power station in-spite of continuous low dose chlorination regime (0.2 ± 0.1 mg L-1) (CLDC), indicating periodical settlement and growth. Continuous arrival of mussels (colonized in the sub seabed tunnel intake section) at the pump house indicated that the mussels were able to tolerate and survive in a chlorinated environment, for varying time periods and were dislodged when they become weak and subsequent death, leading to flushing out of the system. In the present study, effect of continuous chlorination [0.2 mg L-1 (in-plant use); 0.5 mg L-1 (shock dose) & 1.0 mg L-1 (high levels)] was evaluated on mussels to assess; (a) time taken for mortality, (b) action of chlorine on physiological, genetic, metabolic and neuronal processes. 100% mortality of mussels was observed after 15 (0.2 mg L-1); 9 (0.5 mg L-1) and 6 days (1.0 mg L-1) respectively. Extended valve closure due to chlorination resulted in stress, impairing the respiratory and feeding behavior leading to deterioration in mussel health. Pseudofaeces excretion reduced to 68% (0.2 mg L-1); 10% (0.5 mg L-1) and 89% (1.0 mg L-1) compared to controls. Genotoxicity was observed with increase in % tail DNA fraction in all treatments such as 86% (0.2 mg L-1); 76% (0.5 mg L-1) and 85% (1.0 mg L-1). Reactive Oxygen Species (ROS) stress biomarkers increased drastically/peaked within the first 3 days of continuous chlorination with subsequent quenching by antioxidant enzymes. Gill produced highest generation of ROS; 38% (0.2 mg L-1); 97% (0.5 mg L-1); 98% (1.0 mg L-1). Additionally, it was shown that 84% (0.2 mg L-1), 72% (0.5 mg L-1), and 80.4% (1.0 mg L-1) of the neurotransmitter acetylcholinesterase activity was inhibited by chlorine at the nerve synapse. The cumulative impact of ROS generation, neuronal toxicity, and disrupted functions weakens the overall health of green mussels resulting in mortality.

Environmentally realistic concentrations of cocaine in seawater disturbed neuroendrocrine parameters and energy status in the marine mussel Perna perna.

Cocaine (COC) is a powerful illicit drug frequently detected in the aquatic environment. COC acts by inhibiting the reuptake of dopamine (DOPA) and 5-hydroxytryptamine (5-HT - serotonin) and causes endocrine disturbances in mammals. This study investigated similar effects from cocaine exposure in the marine mussel Perna perna, as well as neurotoxicity and energy imbalances. Mussels were exposed to COC (0.2 µg.L-1 and 2 µg.L-1) for periods of 48, 96, and 168 h. Acetylcholinesterase (AChE) was measured in adductor muscle tissue to determine neurotoxicity, and neurotransmitter levels (DOPA and 5-HT), monoamine oxidase (MAO) and cyclooxygenase (COX) activity, and energy status (mitrochondrial electron transport, MET, and total lipids, TLP) were evaluated in the mussels' gonads. COC decreased AChE activity in the mussels exposed to 0.2 µg.L-1 and 2 µg.L -1 after 168 h, and all concentrations of COC increased neurotransmitter levels. Increases in MET (0.2 µg.L -1, for all exposure periods) and TLP (0.2 µg.L 1 after 48 h, and 2 µg.L -1 after 96 h and 168 h) were also observed. No significant change was detected in MAO activity. COC also decreased COX activity in the mussels exposed to 0.2 µg.L -1 (48 h and 96 h) and 2 µg.L -1 (96 h). These results suggest that COC may compromise neurotransmitter levels and COX activity. Furthermore, the changes in MET and LPT suggest that COC affects the energy balance of the mussels, and could negatively affect physiological processes such as metabolism, hormone production, and embryonic development.

Beyond relaxed: magnesium chloride anaesthesia alters the circulatory metabolome of a marine mollusc (Perna canaliculus).

BACKGROUND: The New Zealand Green-lipped mussel industry is well-established providing vastly to aquaculture exports. To assess mussel health and reproduction status, visual examination of organs and/or collection of haemolymph is commonly applied. Anesthetics, such as magnesium chloride (MgCl2) can be utilized to prevent muscle contraction and keep shells open during sampling. The specific effects of muscle relaxing agents on baseline metabolism in invertebrates is unknown, but it is evident that molecular, cellular and physiological parameters are altered with these chemical applications. To this end, metabolomics approaches can help elucidate the effects of relaxing agents for better assessment of their use as a research tool. METHODS: Adult Green-lipped mussels were anaesthetized for 3 h in a MgCl2 bath, whereafter haemolymph samples were collected and analyzed via gas chromatography-mass spectrometry applying methyl chloroformate alkylation derivatization. RESULTS: Anesthetized mussels were characterized as non-responsive to manual manipulation, with open valves, and limited siphoning function. Metabolite profiling revealed significant increases in the abundances of most metabolites with an array of metabolic activities affected, resulting in an energy imbalance driven by anaerobic metabolism with altered amino acids acting as neurotransmitters and osmolytes. CONCLUSION: This research is the first to use a metabolomics approach to identify the metabolic consequences of this commonly used bivalve relaxing technique. Ultimately the use of MgCl2 anesthetization as a sampling strategy should be carefully evaluated and managed when performing metabolomics-related research.

Accumulation and ecotoxicological risk of weathered polyethylene (wPE) microplastics on green mussel (Perna viridis).

Recent studies have shown that organisms including humans are exposed to microplastics directly or indirectly. The present study aims to examine the ingestion of these microplastics and the consequences of the same by studying the accumulation behavior of weathered Polyethylene (wPE) microplastics. The Perna viridis were exposed chronically to three different environmentally relevant concentrations of wPE for 30 days, followed by a one-week depuration phase. There was no mortality observed in the control and exposed groups, but the feeding rate was observed to have substantially decreased in the group exposed to higher concentration (3 µgL-1) of wPE. It was also observed that a higher number of wPE particles accumulated in the intestine of exposed organisms. Interestingly, the present study revealed the presence of the substantial number of wPE particles in exposed organisms, which may adversely affect the internal organs as well as growth and reproduction. This study perceived that accumulation is marginally influenced by size of wPE. Similarly, biomarker analysis showed that wPE exposure significantly altered both the metabolism and histology of the internal organs of the exposed organisms. Overall, the study confirmed that the intestine was the most sensitive organ followed by gills, adductor muscles, and foot tissue adding new insights into the adverse effects of wPE in the marine ecosystem.

Biochemical metal accumulation effects and metalloprotein metal detoxification in environmentally exposed tropical Perna perna mussels.

Marine bivalves have been widely applied as environmental contamination bioindicators, although studies concerning tropical species are less available compared to temperate climate species. Assessments regarding Perna perna mytilid mussels, in particular, are scarce, even though this is an extremely important species in economic terms in tropical countries, such as Brazil. To this end, Perna perna mytilids were sampled from two tropical bays in Southeastern Brazil, one anthropogenically impacted and one previously considered a reference site for metal contamination. Gill metallothionein (MT), reduced glutathione (GSH), carboxylesterase (CarbE) and lipid peroxidation (LPO) were determined by UV-vis spectrophotometry, and metal and metalloid contents were determined by inductively coupled plasma mass spectrometry (ICP-MS). Metalloprotein metal detoxification routes in heat-stable cellular gill fractions were assessed by size exclusion high performance chromatography (SEC-HPLC) coupled to an ICP-MS. Several associations between metals and oxidative stress endpoints were observed at all four sampling sites through a Principal Component Analysis. As, Cd, Ni and Se contents, in particular, seem to directly affect CarbE activity. MT is implicated in playing a dual role in both metal detoxification and radical oxygen species scavenging. Differential SEC-HPLC-ICP-MS metal-binding profiles, and, thus, detoxification mechanisms, were observed, with probable As-, Cu- and Ni-GSH complexation and binding to low molecular weight proteins. Perna perna mussels were proven adequate tropical bioindicators, and further monitoring efforts are recommended, due to lack of data regarding biochemical metal effects in tropical species. Integrated assessments, as performed herein demonstrate, are invaluable in evaluating contaminated aquatic environments, resulting in more accurate ecological risk assessments.

Cinnamaldehyde Could Reduce the Accumulation of Diarrhetic Shellfish Toxins in the Digestive Gland of the Mussel Perna viridis under Laboratory Conditions.

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.

Molecular and biochemical effects of the antifouling DCOIT in the mussel Perna perna.

Biological fouling is an unwanted phenomenon that results in economic losses to the shipping industry. To prevent fouling, antifouling paints are used. DCOIT (4,5- dichloro-2-n-octyl-4-isothiazolin-3-one) is a biocide present in many antifouling paint formulations, and is toxic to a wide range of organisms. The aim of the present study was to evaluate the effects of DCOIT on oxidative stress indicators of the brown mussel, Perna perna. Molecular (SOD-like, GSTO-like and MGST-like mRNA levels) and biochemical (activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), and levels of glutathione (GSH), reactive oxygen species (ROS) and protein carbonyls (PCO)) components were evaluated. Further, levels of biomarkers were assessed in the gills and digestive glands of mussels. Bivalves were exposed to DCOIT (control, 0.1 µg/L and 10 µg/L) for up to 96 h. DCOIT exposure decreased GSH content in gills. Moreover, exposure to DCOIT also decreased CAT activity in the gills and digestive glands of mussels. GST activity increased in digestive gland after exposure for 24 h to both concentrations of DCOIT tested. SOD activity, ROS levels and PCO content were not affected by exposure to the contaminant. Regarding the molecular biomarkers evaluated, DCOIT exposure altered mRNA levels of SOD-like in both tissues after 24 and 96 h of exposure, and decreased MGST-like mRNA levels in the digestive gland after 96 h of exposure to the chemical. These findings suggested that exposure to DCOIT may alter the biochemical and molecular functioning of P. perna, which may harm the species.

Biodegradable chelating agent improves the survival of early larvae for shellfish aquaculture.

The toxicity of heavy metals commonly impacts the survival of crustacean and bivalve larvae in hatchery culture, and this has led to the widespread use of EDTA to decrease this toxicity. Since EDTA has a very poor biodegradability leading to potential persistent environmental effects, alternative methods to prevent heavy metal toxicity to shellfish larvae are needed. EDDS is a biodegradable potential alternative to EDTA for this application and was tested as a treatment of the seawater used for rearing aquaculture Greenshell™ mussel (Perna canaliculus) larval embryos in this study. Mussel embryos reared with EDTA or EDDS had significantly better survival than without. The concentrations and spatial distributions of heavy metals in D-veliger larvae as determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and X-ray Fluorescence Microscopy (XFM) suggested that chelating agents increased the levels of calcium in larvae while they reduced the concentration of zinc. In addition, where decreased accumulation of the other heavy metals was not observed, chelating agents affected their distribution within the larvae, especially for copper and arsenic. This is the first study to test the use of EDDS for aquaculture hatchery application and shows that EDDS is an effective biodegradable alternative to EDTA that can mitigate the effects of heavy metals for shellfish larval rearing.

Biomarker responses in New Zealand green-lipped mussels Perna canaliculus exposed to microplastics and triclosan.

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.

Effects of DCOIT (4,5-dichloro-2-octyl-4-isothiazolin-3-one) to the haemocytes of mussels Perna perna.

Bivalve molluscs rely only on an innate immune system to execute cellular and humoral processes. Haemocytes, the haemolymph circulating cells, play a major role in this type of immunity, principally regarding cellular defences. Considering that environmental pollutants can affect the immune system of invertebrates, this work evaluated the effects of the antifouling biocide 4,5-dicloro-2-n-octil-4-isotiazolin-3-ona (DCOIT) on the haemocytes of mussels Perna perna. Individuals were exposed to 0 (control), 0.1 µg L-1 and 10 µg L-1 of DCOIT for up to 96 h. The analysed parameters included: total (THC) and differential (DHC) haemocyte count, cellular viability, adhesion capacity, phagocytic activity, levels of reactive oxygen species and DNA damage. Moreover, the stress on stress (SOS) response of mussels was analysed as a general stress index. The results show that DCOIT increased the haemocyte adhesion capacity and caused a decrease in THC and in the haemocyte viability after 24 h of exposure. After 96 h of exposure, DCOIT only affected the haemocyte adhesion capacity, which was decreased by biocide exposure. Moreover, exposure to DCOIT for 96 h did not affect the capacity for air survival of mussels. These results indicate that DCOIT interferes in important parameters associated with the innate immunity of P. perna, mainly after 24 h of exposure. It is suggested that the animals were able to develop some compensatory response strategy, making them more resistant to the biocide.

De novo transcriptome analysis of the mussel Perna viridis after exposure to the toxic dinoflagellate Prorocentrum lima.

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.

Effects of chlorothalonil on the antioxidant defense system of mussels Perna perna.

Chlorothalonil is an effective fungicide used in agriculture and formulations of antifouling paints, which use and possible toxicity has been generating great concern. Thus, the present study investigated the effects of chlorothalonil on the antioxidant defense system (ADS) of the mussel Perna perna. The ADS was evaluated in gills and digestive gland after 24 h and 96 h of exposure to environmental relevant levels of chlorothalonil (0.1 and 10 µg/L). The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), glutamate cysteine-ligase (GCL) and glutathione S-transferase (GST), levels of non-enzymatic defenses, represented by glutathione (GSH), and lipoperoxidation (LPO) and protein carbonyls (PCO) were evaluated. Results indicated that exposure to chlorothalonil is affecting the ADS in both tissues. While the activity of SOD increased and GST and GSH were not altered in gills, they decreased in digestive gland after 24 h of exposure to 10 µg/L of chlorothalonil. The contrasting results indicate that gills and digestive gland presented different patterns of responses after exposure to chlorothalonil. Moreover, a tissue-specific response to chlorothalonil was observed. Gills could be acting as the first line of defense, presenting higher enzymatic levels with minor effects on the parameters analyzed. On the other hand, digestive gland, with lower levels of antioxidant defenses, was the most affect organ by chlorothalonil. It also should be highlighted that the fungicide reduced the glutathione metabolism in the digestive gland, which can lead to an imbalance of the redox state within the cells of animals.

Effect of sublethal gradient concentrations of nickel on postlarvae of Penaeus monodon, Perna viridis and Terapon jarbua: Enzyme activities and histopathological changes.

The present study evaluates the enzyme activities and histopathological changes in the post larvae (PL) of shrimp (Penaeus monodon), green mussel (Perna viridis) and fingerlings of crescent perch (Terapon jarbua) exposed to sublethal gradient concentrations of Nickel (Ni). The median lethal concentration (LC50) values were 2.49, 66.03 and 43.92 mg Ni L-1 derived for the PL of shrimp, green mussel and fish fingerlings respectively. No Observed Effect Concentration (NOEC), Lowest Observed Effect Concentration (LOEC) and chronic values of the PL of shrimp were 46.5, 73.0 and 58.3 µg Ni L-1 derived for the 21-d survival endpoint. The NOEC, LOEC and chronic values for the 30-d survival endpoint of the green mussels and fish fingerlings were 4.6, 6.32, 5.4 and 1.95, 2.6, 2.25 mg Ni L-1 respectively. The isoforms of esterase, superoxide dismutase and malate dehydrogenase activities in the whole body tissues of test organisms were studied by native polyacrylamide gel electrophoresis after exposure to Ni. Histological examination of compound eye sections of shrimp revealed deformation, compression, fusion and detachement in the corneal cells from the corneal facet of the ommatidia indicating cellular anomalies due to Ni toxicity. Gill sections of the green mussel witnessed reduced haemolymph in sinuses of gill filaments, degenerative changes in interfilamentous junction and necrosis of frontal ciliated epithelial cells with vacuoles after exposure to Ni. Nickel affects the vision of shrimp and fish fingerlings, gills and byssus of green mussels.

High-throughput identification of heavy metal binding proteins from the byssus of chinese green mussel (Perna viridis) by combination of transcriptome and proteome sequencing.

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.

Up-regulation of Nrf2-dependent antioxidant defenses in Perna viridis after exposed to Prorocentrum lima.

It is well documented that diarrhetic shellfish poisoning (DSP) toxins have strong genetic toxicity, cytotoxicity and oxidative damage to bivalve species. However, these toxic effects seem to decrease with the extension of exposure time and the increment of the toxin concentration, the mechanism involved remained unclear, though. In this paper, we found that expression of the genes related to cytoskeleton and Nrf2 signaling pathway displayed different changes over time in the gill of Perna viridis after exposure to DSP toxins-producing microalga Prorocentrum lima. During the short-term exposure (3 h and 6 h), KEAP1 gene expression was significantly up-regulated, coupled with up-regulation of MRP, ABCB1 and CAT transcriptions and down-regulation of GPx1 and NQO1 mRNA. After longer exposure to high density of P. lima, Nrf2 was significantly up-regulated, accompanied with up-regulation of Nrf2 pathway related genes such as NQO1, SOD, GST-ω and ABCB1, whereas KEAP1 was down-regulated. TUBA1C and TUBB1 transcripts were significantly down-regulated after short-term exposure of P. lima, but both of them were up-regulated at 96 h after exposure to high density of P. lima. Paraffin section demonstrated that P. lima had a strong damage on the gill of mussels during the short-term exposure. However, the negative effect to the gill decreased, and the gill restored after longer exposure (96 h). Taking together, we proposed that P. lima had a negative impact on cytoskeleton of mussel gill tissue, could cause oxidative damage to the gills. However, longer exposure of P. lima in high density could activate Nrf2 signaling pathway, thereby reducing the influence of toxin on mussel. Our study might provide a novel clue for the resistance mechanism of shellfish to DSP toxins.

Marine contamination and cytogenotoxic effects of fluoxetine in the tropical brown mussel Perna perna.

Concerns are growing about the presence of fluoxetine (FLX) in environmental matrices, as well as its harmful effects on non-target organisms. FLX in aquatic ecosystems has been detected in a range varying from pg/L to ng/L, while adverse effects have been reported in several organisms inhabiting freshwater and marine environments. The present study quantifies FLX concentrations in seawater samples from Santos Bay, Brazil and assesses metabolic responses and sublethal effects on the tropical brown mussel Perna perna. Levels of ethoxyresorufin­O­deethylase, dibenzylfluorescein dealkylase, glutathione S-transferase, glutathione peroxidase, cholinesterase, lipoperoxidation, and DNA damage were assessed in the gills and digestive gland of these animals, and lysosomal membrane stability was also assessed in hemocytes. FLX altered phase I and II enzyme activities, caused cytogenotoxic effects, and negatively impacted the overall health of mussels exposed to environmentally relevant concentrations. These findings contribute to characterize the risks of introducing this drug into the marine environment.

Responses of CYP450 in the mussel Perna viridis after short-term exposure to the DSP toxins-producing dinoflagellate Prorocentrum lima.

Diarrhetic shellfish poisoning (DSP) toxins are key shellfish toxins that cause diarrhea, vomiting and even tumor. Interestingly, bivalves such as Perna viridis have been reported to exhibit some resistances to alleviate toxic effects of DSP toxins in a species-specific manner. Nevertheless, the molecular mechanisms underlying the resistance phenomenon to DSP toxins, particularly the mechanistic role of CYP450 is scant despite its crucial role in detoxification. Here, we exposed P. viridis to Prorocentrum lima and examined the expression pattern of the CYP450 and our comprehensive analyses revealed that P. lima exposure resulted in unique expression pattern of key CYP450 genes in bivalves. Exposure to P. lima (2 × 105 cells/L) dramatically orchestrated the relative expression of CYP450 genes. CYP2D14-like mRNA was significantly down-regulated at 6 h in gill, but up-regulated at 2 h in digestive gland compared with control counterparts (p < 0.05), while CYP3A4 mRNA was increased at 12 h in gill. After exposure to P. lima at 2 × 106 cells/L, the expression of CYP3A4 mRNA was significantly increased in digestive gland at 2 h and 12 h, while CYP2D14-like was up-regulated at 6 h. Besides, CYP3L3 and CYP2C8 also exhibited differential expression. These data suggested that CYP3A4, CYP2D14-like, and even CYP3L3 and CYP2C8 might be involved in DSP toxins metabolism. Besides, provision of ketoconazole resulted in significant decrement of CYP3A4 in digestive gland at 2 h and 12 h, while the OA content significantly decreased at 2 h and 6 h compared to control group without ketoconazole. These findings indicated that ketoconazole could depress CYP3A4 activity in bivalves thereby altering the metabolic activities of DSP toxins in bivalves, and also provided novel insights into the mechanistic role of CYP3A4 on DSP toxins metabolism in bivalves.

Impacts of the toxic benthic dinoflagellate Prorocentrum lima on the brown mussel Perna perna: Shell-valve closure response, immunology, and histopathology.

Prorocentrum lima is a widely distributed marine benthic dinoflagellate that produces diarrhetic toxins, okadaic acid (OA) and its analogs, that may promote damage on bivalve tissues and cellular responses. Cultivation of the brown mussel Perna perna represents an important economic activity in the tropical and subtropical regions, where mussels may co-occur with P. lima. This study aimed to assess the behavioral, cellular immune responses, and pathological condition of P. perna following a short-term experimental exposure to P. lima. The toxic dinoflagellate treatment was compared to a non-toxic exposure to the chlorophyte Tetraselmis sp. at similar concentrations. The prevalence of pathological conditions and parasites were assessed, and a pathological index was applied by scoring the prevalences into four levels. Reaction time and the number of stimuli necessary for shell-valve closure response significantly increased after 72 h of P. lima exposure. Circulating hemocyte concentration was significantly lower in P. lima exposed mussels than in control mussels at 48- and 96 h of incubation, while hemocyte relative size in exposed mussels was significantly higher than that in control mussels. Comparatively, phagocytic activity and ROS production by hemocytes was significantly higher in mussels exposed to P. lima at 48- and 96 h of incubation, respectively. In addition, exposed mussels significantly presented exacerbated hemocytic infiltration in digestive organs, higher prevalence of moderate to severe atrophy in digestive tubules, and higher pathological index which suggests an impairment of mussel immunologic responses. A lower prevalence of Rickettsia-like organisms (RLOs), trematodes and copepods in P. lima exposed mussels suggests a direct toxic effect of OA on parasites. The exposure of mussels to P. lima is likely to occur frequently and may lead to constraints on mussel behavior, physiology, and pathological condition.

Detoxification, oxidative stress, and cytogenotoxicity of crack cocaine in the brown mussel Perna perna.

The presence of cocaine and its metabolites and by-products has been identified in different aquatic matrices, making crack cocaine the target of recent studies. The aim of this study was to evaluate the sublethal effects of crack on the brown mussel Perna perna. Mussels were exposed to three concentrations of crack cocaine (0.5, 5.0, and 50.0 µg L-1) for 168 h. Gills, digestive glands, and hemolymph were extracted and analyzed after three different exposure times using a suite of biomarkers (EROD, DBF, GST, GPX, LPO, DNA damage, ChE, and lysosomal membrane stability [LMS]). After 48 and 96 h of exposure, EROD, DBF, GST, GPX activities and DNA strand breaks in the gills increased significantly after 48 and 96 h of exposure. Alterations in LMS were also observed in the mussels exposed to all crack concentrations after 96 and 168 h. Our results demonstrated that crack cocaine is metabolized by CYP-like and GST activities in the gills. GPX was not able to prevent primary genetic damage, and cytotoxic effects in the hemocytes were also observed in a dose- and time-dependent response. Our study shows that the introduction of illicit drugs into coastal ecosystems must be considered a threat to marine organisms.

Copper-induced immunomodulation in mussel (Perna canaliculus) haemocytes.

Copper is a common contaminant in aquatic environments, which may cause physiological dysfunction in marine organisms. However, the toxicity mechanisms of copper in marine bivalves is not fully understood. In this study, we applied an integrated approach that combines flow cytometry and Gas Chromatography-Mass Spectrometry (GC-MS)-based metabolomics to characterize cellular and molecular mechanisms of copper immunotoxicity in New Zealand Greenshell™ mussel (Perna canaliculus) haemolymph. Flow cytometric results showed significant increases in haemocyte mortality, production of reactive oxygen species and apoptosis (via alteration of caspase 3/7 and mitochondrial membrane potential) of haemocytes exposed to increasing total concentrations of Cu2+ (62.5, 125.0 and 187.5 µM) compared to a low Cu2+ concentration (25.0 µM) and control (0.0 µM). In addition to flow cytometric data, our metabolomics results showed alterations of 25 metabolites within the metabolite profile of Cu2+-exposed haemolymph (125 µM) compared to those of control samples. Changes in levels of these metabolites may be considered important signatures of oxidative stress (e.g., glutathione) and apoptosis processes (e.g., alanine, glutamic acid). This study provides insights into the cellular and molecular mechanisms of oxidative stress and apoptosis in marine bivalves and highlights the applicability and reliability of metabolomic techniques for immunotoxicological studies in marine organisms.