Integrated transcriptomic and proteomic analyses of the tissues from the digestive gland of Chlamys farreri following cadmium exposure.

OBJECTIVES: Cadmium causes the pollution of marine habitat and Chlamys farreri is an effective concentrator of heavy metals, the aim of this study was to study the response mechanism of C. farreri to cadmium stress at transcriptomic and proteomic levels. METHODS: Transcriptomic analysis based on RNA-sequencing and proteomic analysis based on isobaric tags for relative and absolute quantitation were performed to reveal the molecular response of C. farreri to different concentrations of cadmium (0.1, 0.3, and 1 mg/L). In addition, a protein-protein interaction (PPI) network was constructed based on the Cytoscape tool to identify hub proteins related to the response of C. farreri to cadmium stress. RESULTS: A total of 24 190 unigenes from 58 683 candidates were annotated in known databases. The numbers of the differentially expressed unigenes (DEGs) was different among the three cadmium-treated groups compared with the control group. DEGs were involved in many pathways such as ABC transporters, protein processing in endoplasmic reticulum and endocytosis. A total of 660 proteins were identified, and differentially expressed proteins (DEPs) among different groups were determined. The overlapping DEGs and DEPs were associated with cadmium response. The upregulated unigene0002618 and downregulated unigene0000904 may be more important for the response of C. farreri to cadmium stress. Unigene0009750 was the hub protein in the PPI network with the highest degree of 20. CONCLUSIONS: Our transcriptomic and proteomic analyses elucidated the molecular response of C. farreri to cadmium stress.

Multiple-Biomarker Approach in a Commercial Marine Scallop from San Jose gulf (Patagonia, Argentina) for Health Status Assessment.

The health status of the commercial Tehuelche scallop Aequipecten tehuelchus from San Román and El Riacho in San José gulf (Patagonia, Argentina) was evaluated through biomarkers widely used in ecotoxicological applications. Natural levels of arsenic (As) and cadmium (Cd) were measured to determine their potential relationships with fluctuations of several oxidative stress biomarkers in the scallop. Oxidative biomarkers, such as catalase (CAT), glutathione S-transferase (GST), superoxide dismutase (SOD), metallothioneins (MT), reactive oxygen species (ROS), α-tocopherol (α-T), and lipid peroxidation (LPO) through thiobarbituric acid reactive substances (TBARS) and lipid radical (LR∙), were measured in gills, digestive gland, and muscle of Tehuelche scallop in winter (August 2015) and summer (January 2016). Levels of As and Cd and of most of the biomarkers (SOD, ROS, TBARS, and LR∙) showed strong seasonal variability in the three tissues. In general, the highest values were recorded in digestive gland. The Integrated Biomarker Response index indicated that the most stressed condition of A. tehuelchus was in summer in San Román. Additionally, the Integrated Biomarker Response index showed a strong relationship among tissues and As and Cd accumulation. This kind of approach could be used as an integrated tool to identify the health status of scallop A. tehuelchus from San José gulf.

The molecular mechanism of Nrf2-Keap1 signaling pathway in the antioxidant defense response induced by BaP in the scallop Chlamys farreri.

In this study, we cloned the full-length cDNA of the Kelch-like ECH-associated protein 1 (Keap1) from the scallops Chlamys farreri (C. farreri). Sequences alignment and phylogenetic analysis showed that CfKeap1 was highly specific in the scallops, and the amino acid sequence identity value is closer to that in zebrafish Keap1b and Nothobranchius furzeri Keap1b than Keap1a. The highest transcription level of CfKeap1 expression was detected in the digestive glands. The gene expressions of CfKeap1, NF-E2-related nuclear factor 2 (Nrf2), Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPx) in digestive glands were evaluated by quantitative real-time PCR (qRT-PCR) after being exposed to benzo(a)pyrene (BaP) (0.25, 1and 4 µg/L) for 15 days, which indicated that the activation of Nrf2 and Keap1 expression can be significantly induced under BaP exposure. RNA interference (RNAi) experiments were conducted to examine the expression profiles of CfKeap1, Nrf2, antioxidant genes (Cu/Zn-SOD, CAT and GPx), mitogen-activated protein kinase (MAPKs) and protein kinase C (PKC) signaling pathways key genes in digestive glands and gills when exposed to BaP. Results showed that the mRNA level of CfKeap1 was significantly decreased by 60.69% and59.485%. The changes of CfKeap1 and Nrf2 suggested that the enhancement of Keap1 expression stimulating Nrf2 degradation. Furthermore, the expression of antioxidant genes were consistent with the Nrf2 gene, which suggesting that Nrf2-Keap1 signaling pathway is required for the induction of antioxidant genes. Besides, the changes of PKC, c-Jun N-terminal kinase (JNK) and p38 genes expression suggested that PKC and MAPKs signaling pathways played a synergistic role with Nrf2-Keap1 signaling pathway in the anti-oxidative defense system of bivalve molluscs. In conclusion, these data demonstrated that Keap1 can sense nucleophilic or oxidative stress factors to regulate the Nrf2 signaling pathway together with Cul3-based E3 Ubiquitin Ligase (E3), and the Nrf2-Keap1 signaling pathway played an important role in modulating gene expression of antioxidant enzymes in bivalve mollusks.

Effect of the Algicide Thiazolidinedione 49 on Immune Responses of Bay Scallop Argopecten Irradians.

The thiazolidinedione 49 (TD49) is an effective algaecide against harmful algae; however, its potential effects on the immune function of the edible bay scallop are unclear. Therefore, the present work studied the effects of TD49 on the immune response in bay scallop by evaluating activities of acid phosphatase (ACP), alkaline phosphatase (ALP), and superoxide dismutase (SOD), as well as nitric oxide (NO) levels, total protein content, and expression of immune genes (CTL-6, PGRP, PrxV, MT, and Cu/Zn-SOD) at 3-48 h post-exposure (hpe) to TD49. The activities of ACP and ALP significantly increased in TD49-treated groups at 3-24 hpe, whereas NO levels decreased significantly in 0.58 and 0.68 µM of TD49 at 6-24 hpe, after which the level was similar to that in the untreated control. Moreover, SOD activity significantly increased in all three concentration groups at 3-6 hpe, while it decreased at 12 hpe in the 0.68 µM TD49 treatment group. Notably, total protein content increased with TD49 treatment at each time interval. The results revealed that variable effects on the expression of immune-related genes were observed after treatment with TD49. The findings demonstrate that exposure of scallops to TD49 changes immune responses and expression of immune-related genes. We hypothesize that TD49 may disrupt immune system in bay scallop. The current investigation highlights the potential negative effects of using TD49 as an algaecide on marine economic bivalves to control harmful algal blooms in marine environments.

A multi-CRD C-type lectin gene Cnlec-1 enhance the immunity response in noble scallop Chlamys nobilis with higher carotenoids contents through up-regulating under different immunostimulants.

C-type lectins have a variety of immunological functions in invertebrates. In order to investigate whether C-type lectin gene and carotenoids do have immune influences on noble scallop Chlamys nobilis under pathogen stress, acute challenges lasting 48 h to Vibrio parahaemolyticus, lipopolysaccharide (LPS), polyinosinic polycytidylic acid (Poly I: C), and PBS were conducted in noble scallop with different carotenoids content. A multi-CRD C-type lectin gene called Cnlec-1 was cloned and its transcripts under different challenges were determined. Full length cDNA of Cnlec-1 is 2267 bp with an open reading frame (ORF) of 1845 bp encoding 614 deduced amino acids, containing four carbohydrate recognition domains (CRD1, CRD2, CRD3 and CRD4). Phylogenetic tree analysis showed that CRDs of Cnlec-1 were clustered with CRDs of shellfish C-type lectins, especially closely related to Chlamys farreri and Argopecten irradians CRDs. Cnlec-1 transcripts were detected in hemocytes, mantle, gonad, kidney, intestines, gill and adductor. Compared with PBS control group, Cnlec-1 transcripts were up-regulated in V. parahaemolyticus, LPS and Poly I: C groups. Furthermore, Cnlec-1 transcript levels of Golden scallops were significantly higher than that of Brown ones at 3-48 h (P < 0.05) in V. parahemolyticus groups, at 24 h in LPS groups and at 12-24 h in Poly I: C groups. These results suggesting that Cnlec-1 is an important immune factor involved in the defense against pathogens in the noble scallop, and carotenoids can enhance the immunity of noble scallop through up-regulating Cnlec-1 to different immunostimulants.

Genome-wide identification, characterization and expression analyses of TLRs in Yesso scallop (Patinopecten yessoensis) provide insight into the disparity of responses to acidifying exposure in bivalves.

Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing specific pathogen-associated molecular patterns, including lipoproteins, lipopeptides, lipopolysaccharide, flagellin, dsRNA, ssRNA and CpG DNA motifs. Although significant effects of TLRs on immunity have been reported in most vertebrates and some invertebrates, the complete TLR superfamily has not been systematically characterized in scallops. In this study, 18 TLR genes were identified from Yesso scallop (Patinopecten yessoensis) using whole-genome scanning. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of the 18 genes. Extensive expansion of TLR genes from the Yesso scallop genome indicated gene duplication events. In addition, expression profiling of PyTLRs was performed at different acidifying exposure levels (pH = 6.50, 7.50) with different challenge durations (3, 6, 12 and 24 h) via in silico analysis using transcriptome and genome databases. Our results confirmed the inducible expression patterns of PyTLRs under acidifying exposure, and the responses to immune stress may have arisen through adaptive recruitment of tandem duplications of TLR genes. Collectively, this study provides novel insight into PyTLRs as well as the specific role and response of TLR signaling pathways in host immune responses against acidifying exposure in bivalves.

Expression profiles of different glutathione S-transferase isoforms in scallop Chlamys farreri exposed to benzo[a]pyrene and chrysene in combination and alone.

Aquatic organisms are increasingly exposed to polycyclic aromatic hydrocarbons (PAHs) due to anthropogenic pressure. This study aimed at evaluating the response of Glutathione S-transferases (GSTs) in scallop Chlamys farreri against benzo[a]pyrene (BaP) and chrysene (CHR) exposure under laboratory conditions. Nine published GST genes were classified into six subfamilies and a new member of rho family was identified for the first time. Twelve GSTs (including nine published GST genes and three in transcriptome established by our laboratory) mRNA transcript levels in the gills, digestive glands, adductor muscle, mantle, testis, ovaries, blood cells of scallops were measured by real-time PCR. The results showed that the mRNA transcript levels of twelve GSTs, except GST-zeta, GST-mu and GST-microsomal, were highest in digestive gland. Accordingly, the mRNA expression levels of GSTs were measured in digestive glands of scallops exposed to BaP (0.1µg/L and 1µg/L), CHR (0.1µg/L and 1µg/L) and their mixtures (0.1µg/L BaP +0.1µg/L CHR and 1µg/L BaP +1µg/L CHR). The results indicated that different GST had specific response to different pollution exposure. In BaP exposure experiment, the mRNA expression level of GST-theta was a potential suitable biomarker. GST-sigma-2 and GST-3, which belonged to sigma class, were sensitive to CHR exposure while GST-microsomal was considered a potential ideal bioindicator to joint exposure of BaP and CHR. In summary, this study investigated the classification of GSTs and provided information about the expression profiles of different class GSTs after PAHs exposure.

Are CuO nanoparticles effects on hemocytes of the marine scallop (Chlamys farreri) caused by particles and/or corresponding released ions?

Manufactured nanoparticles (NPs) have become emerging pollutants and attracted extensive concern about their potential effects on the marine environment. However, the contribution of particles and their corresponding released ions to the overall toxicity of CuO NPs is poorly understood. In this study, we investigated the toxicological effects of CuO NPs and their corresponding released ions on the hemocytes of Chlamys farreri. Both copper species induced membrane damage, and increased lysosome contents in hemocytes. Based on the integrated biomarker responses method, the relative contributions of particles (NPparticle) and dissolved ions (NPion) to the toxicity of CuO NPs after 2h of exposure were 62.07% and 37.93%, respectively, indicating that the particles rather than the dissolved ions were the dominant source of NP toxicity. Transmission/scanning electron microscopy analysis confirmed the greater histopathological effects exerted by particles than Cu ions. Higher reactive oxygen species (ROS) generation induced by NPparticle than by NPion suggested that the intracellular ROS production might be responsible for the NP toxicity. Our findings suggest that particles effects play a key role in risk assessment of CuO NPs on the marine ecosystem.

A multi-biomarker approach in scallop Chlamys farreri to assess the impact of contaminants in Qingdao coastal area of China.

A multi-biomarker approach was carried out to classify the environmental quality and the adverse effects of contaminants on scallop Chlamys farreri. The scallops were collected from three sampling stations in Qingdao coastal area of China in March, May, August and October of 2015. A suite of environmental factors and biomarkers, including temperature, salinity, pH, the concentrations of polycyclic aromatic hydrocarbons (PAHs), tetrabromobisphenol A (TBBPA) and metals (Cr, Mn, Cu, Zn, Cd, Pb, As) in seawater and soft tissue, mRNA expression of aryl hydrocarbon receptor (AhR) and P-glycoprotein (P-gp), 7-ethoxyresorufin O-deethylase (EROD), glutathione-S-transferase (GST), uridine-diphosphate-glucuronyl-transferase (UGT), sulfotransferase (SULT), metallothionein (MT), Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), lipid peroxidation (LPO) and protein carbonyl (PC) contents and DNA strand breaks, were measured in the gill and digestive gland. The results showed that S2 was the most polluted while S1 was identified the least polluted. Despite the differentiation of pollution levels and environmental parameters the selected biomarkers responded efficiently to contaminants. Principal component analysis (PCA) revealed that EROD for PAHs, AhR for TBBPA, MT for Cr, Pb and Mn, LPO and PC for Zn were the effective biomarkers respectively. This study demonstrated that the application of multi-biomarker approach in conjunction with the traditional analysis of environmental parameters and contaminants provided valuable information in environmental risk assessment.

Embryo-larvae and juvenile toxicity of Pb and Cd in Northern Chilean scallop Argopecten purpuratus.

This study aimed to investigate the effects of Cd and Pb on earlier stage of the commercially important scallop (Argopecten purpuratus) in the contamination context of Northern Chile where this specie is farmed, through acute exposure bioassays in embryo-larvae measuring cumulative abnormality (EC50) and juvenile cumulative mortality (LC50) as endpoints, based on environmentally detected concentrations and available toxicological data from similar species. Embryo-larvae exposure indicates 48 h EC50 of 1.55 mg/L Cd, and 0.044 mg/L Pb. On the contrary, 96 h LC50 in juvenile scallops was 0.48 mg/L Cd and 1.47 mg/L Pb. Our results demonstrated differential toxicity between embryo and juvenile scallops that might relate to different primary defense mechanisms or effect in morphological development of individuals in each ontogenetic stage. Compared to similar bivalve metal toxicity tests, this study demonstrated that A. purpuratus embryos are more sensitive to Pb than most other bivalve species. Our results indicate that maximum permitted levels of Pb in marine waters and estuaries (according to Chilean regulation) could pose a risk for scallops' first stage of life (embryo-larvae) development, and needs to be reviewed. Furthermore, Chilean environmental regulations do not have quality standards for marine sediments (currently under discussion), where high levels of metals have been continuously reported.

Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

Immune response of the bay scallop, Argopecten irradians, after exposure to the algicide palmitoleic acid.

Palmitoleic acid (PA) is an effective algicide against the toxin-producing dinoflagellate Alexandrium tamarense; however, its effects on the immune system of the edible bay scallop Argopecten irradians are unclear. Therefore, we investigated the effects of PA on the immune response in A. irradians by assessing total haemocyte counts (THC), alkaline phosphatase activity (ALP), nitrite oxide (NO), glutathione (GSH), and lactate dehydrogenase (LDH) levels, as well as the expression of immune-related genes (FREP, PGRP, HSP90, MnSOD, and Cu/ZnSOD) at various hours post-exposure (hpe) to the compound. THC decreased in PA-treated groups, whereas ALP increased significantly in all of the PA treatment groups at 3 hpe, after which it significantly decreased. The LDH and NO levels were significantly enhanced in the high and medium concentration group. Notably, the GSH level increased in all PA treatment groups at each time interval. Our study revealed that after treatment with different concentrations of PA, variable effects on the expression of genes involved in the immune system response were observed. The results of our study demonstrate that immersing scallops in PA at effective concentrations could result in differential effects on immune system responses and expression of immune-related genes. Specifically, PA may disrupt the endocrine system or affect signal transduction pathways in the scallops. Therefore, the present study highlights the potential risk of using the PA as an algicide to control algal bloom outbreaks in the marine environment.

Effects of pH and bicarbonate on mitochondrial functions of marine bivalves.

Estuarine organisms including mollusks are exposed to periodic oxygen deficiency (hypoxia) that leads to a decrease in intracellular pH and accumulation of bicarbonate (HCO3(-)). These changes can affect cellular bioenergetics; however, their effects on mitochondria of estuarine mollusks are not well understood. We determined the interactive effects of bicarbonate (0-10mM) and pH (7.2 and 6.5) on mitochondrial oxygen consumption (MO2), membrane potential (Δψ) and production of reactive oxygen species (ROS) in two common estuarine bivalves - hard clams Mercenaria mercenaria, and bay scallops Argopecten irradians. In both species, elevated HCO3(-) levels suppressed ADP-stimulated (state 3) MO2 but had little effect on the resting (state 4) respiration. These effects were not mediated by the soluble adenylyl cyclase or cyclic AMP. Effects of the low pH (6.5) on mitochondrial traits were species-specific and depended on the substrate oxidized by the mitochondria. Mild acidosis (pH6.5) had minimal effects on MO2 and Δψ of the bivalve mitochondria oxidizing pyruvate but led to increased rates of ROS production in clams (ROS production could not be measured in scallops). In succinate-respiring mitochondria of clams, mild acidosis suppressed MO2 and increased mitochondrial coupling, while in scallop mitochondria the effects of low pH were opposite. Suppression of mitochondrial oxidative phosphorylation by bicarbonate and/or acidosis may contribute to the metabolic rate depression during shell closure or environmental hypoxia/hypercapnia. These findings have implications for understanding the physiological mechanisms involved in regulation of mitochondrial bioenergetics during hypoxia exposure in estuarine bivalves.

Biological effects of mechanically and chemically dispersed oil on the Icelandic scallop (Chlamys islandica).

This study aimed to simulate conditions in which dispersant (Dasic NS) might be used to combat an oil spill in coastal sub-Arctic water of limited depth and water exchange in order to produce input data for Net Environmental Benefit Analysis (NEBA) of Arctic and sub-Arctic coastal areas. Concentration dependent differences in acute responses and long-term effects of a 48h acute exposure to dispersed oil, with and without the application of a chemical dispersant, were assessed on the Arctic filter feeding bivalve Chlamys islandica. Icelandic scallops were exposed for 48h to a range of spiked concentrations of mechanically and chemically dispersed oil. Short-term effects were assessed in terms of lysosomal membrane stability, superoxide dismutase, catalase, gluthatione S-transferases, glutathione peroxidases, glutathione reductase, glutathione, total oxyradical scavenging capacity, lipid peroxidation and peroxisomal proliferation. Post-exposure survival, growth and reproductive investment were followed for 2 months to evaluate any long-term consequence. Generally, similar effects were observed in scallops exposed to mechanically and chemically dispersed oil. Limited short-term effects were observed after 48h, suggesting that a different timing would be required for measuring the possible onset of such effects. There was a concentration dependent increase in cumulative post-exposure mortality, but long-term effects on gonadosomatic index, somatic growth/condition factor did not differ among treatments.

Effect of the Algaecide Palmitoleic Acid on the Immune Function of the Bay Scallop Argopecten irradians.

Palmitoleic acid (PA), an algicidal compound, is used against the toxin producing dinofagelate Alexandrium tamarense, however, its impact on the edible bay scallop (Argopecten irradians) is still unclear. Therefore, we investigated the impacts of effective algicidal concentrations (20, 40, and 80 mg/L) of PA on immune responses in A. irradians. Various immune parameters including acid phosphatase (ACP) activity, superoxide dismutase (SOD), lysozyme, phagocytic activity, total protein, malondialdehyde (MDA) level, and reactive oxygen species (ROS) production and the expression of immune-related genes (PrxV, CLT-6, MT, and BD) were measured at 3, 6, 12, 24, and 48 h post-exposure (hpe) to PA. Lysozyme activity was lower in scallops at 12-48 hpe to 80 mg/L. SOD, ACP activity, ROS production, the total protein, and MDA level was higher at 12 to 48 hpe with different concentrations of PA. Phagocytic activity increased at 6-12 hpe to 40-80 mg/L of PA, but decreased at 24-48 hpe. The expressions of genes PrxV, CLT-6, MT and BD down-regulated at 3 hpe were observed, while differential expressions from 6-48 hpe with different concentrations of PA. The present study demonstrated that immersing A. irradians in PA at effective concentrations could result in differential effects on non-specific immune responses and expressions of immune-related genes.

The transcriptomic response to copper exposure in the digestive gland of Japanese scallops (Mizuhopecten yessoensis).

The present study was conducted to elucidate the effects of copper exposure on the immune system and lipid metabolism of the Japanese scallop, Mizuhopecten yessoensis. Transcriptional levels of differentially expressed genes (DEGs)in M. yessoensis digestive gland tissue were analyzed using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 841 and 877 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 3 gene ontology terms and 15 pathways involved in the response to copper exposure. Analysis of transcripts related to the immune response revealed a complex pattern of innate recognition receptors, including toll-like receptors, NOD-like receptors and downstream pathway effectors, including those involved in apoptosis. Furthermore, genomic analysis revealed that genes involved in extracellular matrix (ECM)-receptor interactions were enriched in Cu-exposed scallop glands. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers for the aquaculture of M. yessoensis.

Modulation of pumping rate by two species of marine bivalve molluscs in response to neurotransmitters: Comparison of in vitro and in vivo results.

Most studies regarding the neuroanatomy and neurophysiology of molluscan ctenidia have focused on isolated ctenidial tissue preparations. This study investigated how bivalve molluscs modulate their feeding rates by examining the effects of a variety of neurotransmitters, including serotonin, dopamine, and the dopamine agonist apomorphine on both isolated ctenidial tissue and in intact members of two commercially important bivalve species: the blue mussel, Mytilus edulis; and the bay scallop Argopecten irradians. In particular, we examined the effect of changes in: 1) beat of the lateral cilia (in vitro), 2) distance between ctenidial filaments and/or plicae (in vivo), and 3) diameter of the siphonal openings (in vivo) on alteration of bulk water flow through the mantle cavity. Important differences were found between isolated tissue and whole animals, and between species. Drugs that stimulated ciliary beat in vitro did not increase water processing rate in vivo. None of the treatments increased water flow through the mantle cavity of intact animals. Results suggest that A. irradians was primarily modulating lateral ciliary activity, while M. edulis appeared to have a number of ways to control water processing activity, signifying that the two species may have different compensatory and regulatory mechanisms controlling feeding activity.

Environmentally relevant concentrations of benzo[a]pyrene affect steroid levels and affect gonad of male scallop Chlamys farreri.

Sexually mature male Chlamys farreri were exposed to benzo[a]pyrene for 10 days at four concentrations at 0, 0.025, 0.5 and 10 µg/L. Fluctuations in sex steroids during the exposure period were observed, which indicated that sex steroids have a role in gamete development. Exposure to B[a]P altered levels of 17ß-estradiol, testosterone, and progesterone. Furthermore, B[a]P treatments induced oxidative stress in a dose-dependent manner on spermary of scallop and led to delayed development and damaged spermatid in germinal epithelium of spermary. Overall, B[a]P affected the steroid levels and induced gonadal toxicity at environmentally relevant concentrations.

Acetylcholine modulates the immune response in Zhikong scallop Chlamys farreri.

Acetylcholine (ACh) is an indispensable neurotransmitter and neuromodulator in the cholinergic nervous system and it is implicated in the dynamic modulation of immune response in vertebrates. Although ACh has also been identified in most invertebrates, the knowledge about its immunomodulation is still limited. In the present study, the immunoreactivities of ACh and acetylcholinesterase (AChE) were observed in all the tested tissues of scallop Chlamys farreri, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. The ACh concentration in the supernate of scallop hemolymph increased from 11.59 ± 0.27 to 14.36 ± 0.17 µM L(-1) at 6 h after LPS (0.5 mg ml(-1)) stimulation, and increased to 15.51 ± 1.20 µM L(-1) at 3 h after the stimulation of tumor necrosis factor alpha (TNF-α) (50 ng ml(-1)). After LPS stimulation, the mRNA expression levels of superoxide dismutase (CfSOD), catalase (CfCAT) and lysozyme (CfLYZ) in hemocytes increased significantly at 3 h (P < 0.05), 6 h (P < 0.05) and 12 h (P < 0.05), respectively. Compared with the LPS treatment, the induction of CfSOD, CfCAT and CfLYZ expression in hemocytes was repressed effectively (P < 0.05) by the co-stimulation of LPS and ACh (5 × 10(-7) M) at 3 h (P < 0.05), 6 h (P < 0.05) and 12 h (P < 0.05), respectively. Furthermore, the expression level of CfCAT in hemocytes increased significantly after 12 h by the co-stimulation with LPS and ACh (P < 0.05). These results indicated collectively that the scallop cholinergic nervous system could be activated by immune stimulations, and it might play an essential role in immunomodulation of scallops.

The transcriptomic response to copper exposure by the gill tissue of Japanese scallops (Mizuhopecten yessoensis) using deep-sequencing technology.

The bivalve Mizuhopecten yessoensis has been greatly impacted by marine pollutants in northern China. To elucidate the toxicological mechanism of copper exposure on the immune system, we investigated differentially expressed genes (DEGs) and transcript abundance in M. yessoensis gill tissue using the deep-sequencing platform Illumina HiSeq™ 2000. In total, 1312 and 2237 genes were identified as significantly up- or down-regulated, respectively. In addition, significant enrichment analysis identified 9 GO terms and 38 pathways involved in the response to copper exposure. The analysis of immune-related transcripts revealed a complex repertoire of innate recognition receptors, including toll-like receptors, NOD-like receptors and RIG-like receptors. Downstream pathway effectors, such as apoptotic, lysosomal and C-type lectin transcripts, were also analyzed. These results will provide a resource for subsequent gene expression studies regarding heavy metal exposure and the identification of copper-sensitive biomarkers to monitor the aquaculture of M. yessoensis.