Enzymes and non-enzymatic antioxidants responses to sequential cold stress in polymorphic noble scallop Chlamys nobilis with different total carotenoids content.

Noble scallop, an economically important edible marine bivalve displays polymorphism in shells (golden and brown) and flesh colors (orange and white). Mass mortality of noble scallops usually occurs during the winter months. Interestingly, carotenoid-rich golden scallops demonstrated much higher survival rates than brown scallops in winter. In order to understand the response of polymorphic noble scallops to sequential cold stress, the present study aimed to investigate the enzyme and non-enzymatic antioxidant responses of golden and brown scallops under sequential cold stress. Parameters evaluated included total carotenoid content (TCC), fatty acid composition, total antioxidant capacity (TAC), methylenedioxyamphetamine (MDA) content, catalase (CAT) activity, and superoxide dismutase (SOD) enzyme activity. The results of the present study revealed that golden scallops have higher cold tolerance than brown scallops. Golden and brown scallops are well adapted to low water temperature of above 12 °C, but in areas where winter water temperatures are below 12 °C, golden scallops are more suitable for aquaculture than brown scallops. The findings of this study are crucial to understanding the physiological responses of polymorphic scallops to cold stress and identify suitable candidates for winter aquaculture.

Differential responses of a pi-class glutathione S-transferase (CnGSTp) expression and antioxidant status between golden and brown noble scallops under pathogenic stress.

Glutathione S-transferases (GSTs) play important roles in immunity by protecting organisms against the damage of reactive oxygen species (ROS). In this study, a pi-class GST cDNA sequence was first cloned from noble scallop Chlamys nobilis (named CnGSTp). The full length cDNA of CnGSTp was 922 bp, encoding a cytosolic protein of 202 amino acids residues, with predicted molecular masses of 23.1 kDa. Then an acute Vibrio Parahaemolyticus challenge experiment was conducted by using the Golden and Brown noble scallops with different total carotenoids content (TCC), and CnGSTp expression level, TCC and ROS level was separately determined. The results showed that ROS and CnGSTp expression levels were significantly up-regulate under Vibrio Parahaemolyticus challenge than the control group (P < 0.05). The Golden scallops showed significantly higher CnGSTp expression level and lower ROS level in hemocytes than the Brown ones (P < 0.05). Moreover, there is a significantly positive correlation between TCC and ROS in the Golden scallops. The present results revealed that CnGSTp plays important roles in immune response and carotenoids play assistant roles in antioxidant defense system under pathogenic stress in the noble scallop.

The mechanism of Mitogen-Activated Protein Kinases to mediate apoptosis and immunotoxicity induced by Benzo[a]pyrene on hemocytes of scallop Chlamys farreri in vitro.

Benzo [a]pyrene (B [a]P) has received widespread attention for serious pollution in the sea, which may reduce immunity and lead to the outbreak of disease in bivalves. However, the mechanism of immunotoxicity induced by B [a]P in bivalves was still unclear. Previous studies have found that Mitogen-Activated Protein Kinases (MAPKs) including three classic pathways (ERK, p38 and JNK) play an important role in mediating this process. Thus, in order to explore the mechanism of immunotoxicity induced by B [a]P in scallop Chlamys farreri, hemocytes were treated with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) for 1 h and then incubation with B [a]P for 24 h at 1 µg/mL. Indexes including oxidative damage, apoptotic rate, and immune indicators were detected in the present study. The results showed that the increase of Reactive Oxygen Species (ROS) and DNA damage induced by B [a]P was inhibited with PD98059 and SB203580. Besides, lysosomal membrane stability (LMS) damage was promoted by PD98059, while it was opposite when treated with SB203580. Moreover, the ascended apoptosis rate induced by B [a]P was increased significantly after treatment with PD98059, but it was remarkably attenuated by SB203580 and SP600125. However, the opposite pattern was showed in phagocytosis compared with apoptosis rate in all of three inhibitors. In addition, antibacterial activity and bacteriolytic activity were enhanced by SB203580 while inhibited by PD98059. Therefore, these results showed that MAPKs directly or indirectly mediate the decrease of oxidative damage, apoptosis and immune defense ability of C. farreri hemocytes, which suggesting ERK/p38/JNK pathways have different functions in the apoptosis and immunity of C. farreri hemocytes after B [a]P exposure. In conclusion, this study intended to enrich the theoretical basis for immunotoxicology of bivalves exposed to pollutants.

Biochemical Characterization of a Novel Endo-1,3-ß-Glucanase from the Scallop Chlamys farreri.

Endo-1,3-ß-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-ß-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-ß-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-ß-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-ß-glucanases, which facilitated development of a unique endo-1,3-ß-glucanase tool in the synthesis of novel glycosides.

A mitochondrial manganese superoxide dismutase involved in innate immunity is essential for the survival of Chlamys farreri.

Superoxide dismutase (SOD) ubiquitously found in both prokaryotes and eukaryotes functions as the first and essential enzyme in the antioxidant system. In the present study, a manganese SOD (designated as CfmtMnSOD) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfmtMnSOD contained a 681 bp open reading frame (ORF), encoding a peptide of 226 amino acids. A SOD_Fe_N domain and a SOD_Fe_C domain were found in the deduced amino acid sequence of CfmtMnSOD. The mRNA transcripts of CfmtMnSOD were constitutively expressed in all the tested tissues, including gill, gonad, hepatopancreas, hemocytes, mantle and muscle, with the highest expression level in hemocytes. After the stimulation of Vibrio splendidus, Staphylococcus aureus and Yarrowia lipolytica, the mRNA transcripts of CfmtMnSOD in hemocytes all significantly increased. The purified rCfmtMnSOD protein exhibited Mn2+ dependent specific and low stable enzymatic activities. After Vibrio challenge, the cumulative mortality of CfmtMnSOD-suppressed scallops was significantly higher than those of control groups and the semi-lethal time for CfmtMnSOD-suppressed scallops was rather shorter than those of control groups either. Moreover, the final mortality rate of CfmtMnSOD-suppressed group was significant higher than those of control groups, even without Vibrio challenge. All these results indicated that CfmtMnSOD was efficient antioxidant enzyme involved in the innate immunity, and also essential for the survival of C. farreri.

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.

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.

Characterization of three mitogen-activated protein kinases (MAPK) genes reveals involvement of ERK and JNK, not p38 in defense against bacterial infection in Yesso scallop Patinopecten yessoensis.

Mitogen-activated protein kinases (MAPKs) are protein Ser/Thr kinases that play a vital role in innate immune responses by converting extracellular stimuli into a wide range of cellular responses. Although MAPKs have been extensively studied in various vertebrates and invertebrates, our current understanding of MAPK signaling cascade in scallop is in its infancy. In this study, three MAPK genes (PyERK, PyJNK, and Pyp38) were identified from Yesso scallop Patinopecten yessoensis. The open reading frame of PyERK, PyJNK, and Pyp38 was 1104, 1227, and 1104 bp, encoding 367, 408, and 367 amino acids, respectively. Conservation in some splicing sites was revealed across the three PyMAPKs, suggesting the common descent of MAPKs genes. The expression profiles of PyMAPKs over the course of ten different developmental stages showed that they had different expression patterns. In adult scallops, PyMAPKs were primarily expressed in muscles, hemocytes, gill, and mantle. To gain insights into their role in innate immunity, we investigated their expression profiles after infection with Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Vibrio anguillarum). Significant difference in gene expression was only found in PyERK and PyJNK, but not Pyp38, suggesting Pyp38 may not participate in immune response to bacterial infection. Besides, PyERK and PyJNK exhibited more drastic change against the invasion of V. anguillarum than M. luteus, suggesting they could be more sensitive to Gram-negative bacteria than Gram-positive bacteria. This study provides valuable resource for elucidating the role of MAPK signal pathway in bivalve innate immune response.

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.

The comparative proteomics analysis revealed the modulation of inducible nitric oxide on the immune response of scallop Chlamys farreri.

Nitric oxide (NO) is an important gasotransmitter which plays a key role on the modulation of immune response in all vertebrates and invertebrates. In the present study, the modulation of inducible NO on immune response of scallop Chlamys farreri was investigated via proteomic analysis. Total proteins from hepatopancreas of scallops treated with lipopolysaccharide (LPS) and/or the inhibitor of vertebrate inducible NO synthase (S-methylisothiourea sulfate, SMT) for 12 h were analyzed via 2-D PAGE and ImageMaster 2D Platinum. There were 890, 1189 and 1046 protein spots detected in the groups treated by phosphate buffered saline (PBS), LPS and LPS+SMT, respectively, and 26 differentially expressed protein spots were identified among them. These proteins were annotated with binding or catalytic activity, and most of them were involved in metabolic or cellular processes. Some immune-related or antioxidant-related molecules such as single Ig IL-1-related receptor, guanine nucleotide-binding protein subunit beta-like protein and peroxiredoxin were identified, and the changes of their expression levels in LPS group were intensified significantly after adding SMT. The decreased expression level of tyrosinase and increased level of glutathione S-transferase 4 in LPS group were diametrically reversed by appending SMT. Moreover, interferon stimulated exonuclease gene 20-like protein and copper chaperone for superoxide dismutase were only induced by LPS+SMT stimulation but not by LPS stimulation. These data indicated that NO could modulate many immunity processes in scallop, such as NF-κB transactivation, cytoskeleton reorganization and other pivotal processes, and it was also involved in the energy metabolism, posttranslational modification, detoxification and redox balance during the immune response.

Functional characterization of a Δ5-like fatty acyl desaturase and its expression during early embryogenesis in the noble scallop Chlamys nobilis Reeve.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential in lots of important physiological processes, while, many marine species have no or limited ability of endogenous PUFA biosynthesis, normally due to the lack of key enzymes such as fatty acid desaturase (FAD). In this study, we isolated a scallop Chlamys nobilis cDNA with high homology to vertebrate FADs. Functional characterization in recombinant yeast Saccharomyces cerevisiae showed that scallop FAD exhibited Δ5-desaturation activity towards both saturated and PUFA substrates. Thus, it efficiently desaturated exogenously added PUFA C20:4(n - 3) and C20:3(n - 6) to C20:5(n - 3) (EPA) and C20:4(n - 6) (ARA) respectively. It also converted the yeast's endogenous C18:0 into C18:1(n - 13), and participated in the biosynthesis of non-methylene-interrupted FA by introducing a double bond to C20:3(n - 3) and C20:2(n - 6) in the Δ5 carbon. Temporal transcript profile of scallop FAD was studied during early embryonic development. High level of mRNA was found at the beginning of embryogenesis (egg) and noticeable decreases of were observed during larvae development, suggesting maternal FAD mRNA transfer to the embryo. Further, FAD transcripts were detected in all tissues analyzed, with the gonad and hepatopancreas showing the highest expression.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri.

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot(-1), 0.40 ± 0.06 U mgprot(-1), P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot(-1), 93.0 ± 6.3 nmol mgprot(-1), P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

Phenoloxidase in the scallop Chlamys farreri: purification and antibacterial activity of its reaction products generated in vitro.

Phenoloxidase (PO) was purified from hemocytes of the scallop Chlamys farreri using native-PAGE and gel permeation column chromatography, and then substrate specificity and antibacterial activity generated from reaction products of purified PO were analyzed. The results showed purified PO had a molecular mass of 576 kDa in native-PAGE and 53 kDa in denatured PAGE, and could catalyze the substrates L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine, catechol and hydroquinone suggesting it is a type of p-diphenoloxidase. Using dopamine as a substrate, PO reaction products significantly inhibited the growth of Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas salmonicida. No significant inhibition was found in Streptococcus dysgalactiae, Streptococcus iniae, Micrococcus lysodeikticus and Edwardsiella tarda. When L-DOPA was used as a substrate, significant inhibition occurred in A. salmonicida only.

The expression of dopa decarboxylase and dopamine beta hydroxylase and their responding to bacterial challenge during the ontogenesis of scallop Chlamys farreri.

Dopa decarboxylase (DDC) and dopamine beta hydroxylase (DBH) is responsible for the synthesis of dopamine and norepinephrine, respectively. In the present study, dopa decarboxylase (CfDDC) and dopamine beta hydroxylase (CfDBH) were selected as indicator to investigate the development of catecholaminergic nervous system in the larvae of scallop Chlamys farreri. The CfDDC and CfDBH transcripts were all detectable during the whole ontogenesis expect for the CfDDC transcripts in 2-cell embryos stage. The expression level of CfDDC and CfDBH mRNA increased significantly in the veliger stage, and reached the peak in late (35.64-fold, P < 0.05) and mid-veliger (400.21-fold, P < 0.05) larvae, respectively. By immunofluorescence, two CfDDC immunoreactive areas were observed in the trochophore and D-hinged larvae, and then three CfDDC immunoreactive areas and two immunopositive fibres formed in early and late veliger larvae, respectively. Two CfDBH immunopositive fibers appeared initially in the early D-hinged stage, and another two similar fibers developed in the late D-hinged stage. The bacteria Vibrio anguillarum challenge could induce the mRNA expression of CfDDC and CfDBH in different developmental stage. The significantly increase of CfDDC mRNA was observed in the trochophore larvae at 12 h (8.61-fold, P < 0.05) and in late D-hinged larvae at 24 h (1.56-fold, P < 0.05) post challenge. The expression level of CfDBH mRNA decreased significantly in late D-hinged larvae at 6 h (0.45-fold, P < 0.05), whereas it increased significantly in late veliger larvae at 12 h after bacterial challenge (14.52-fold, P < 0.05). These results concluded that the scallop catecholaminergic nervous system appeared firstly as the form of dopaminergic neurons in the trochophore larvae, and the developing catecholaminergic nervous system in the trochophore, D-hinged and veliger larvae of scallop could respond to the immune stimulation in different patterns.

An iodothyronine deiodinase from Chlamys farreri and its induced mRNA expression after LPS stimulation.

Iodothyronine deiodinase is responsible for the deiodination of thyroxine T4 to T3, and involved in the complex neuroendocrine-immune regulatory network to optimize the immune response in vertebrate. In this study, the full-length cDNA of an iodothyronine deiodinase (designated as CfDx) was cloned from scallop Chlamys farreri. The complete cDNA sequence of CfDx was of 1404 bp and contained an open reading frame of 900 bp encoding a polypeptide of 299 amino acids. The deduced amino acid sequence of CfDx contained an in-frame TGA stop codon probably encoding an essential selenocysteine (SeC), and there was a conserved region of about 15 amino acids surrounding the SeC residue. The CfDx mRNA transcripts were detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, gonad, gill and mantle, with the higher expression level in hepatopancreas and kidney. After LPS stimulation, the CfDx mRNA expression level in haemocytes increased significantly at 12 h (25.35-fold, P < 0.05) and 24 h (7.62-fold, P < 0.05), and the concentration of T3 in haemolymph increased significantly at 12 h (3.62 ng dL(-1), P < 0.05) even the concentration of T4 did not change significantly. After scallop received an injection of 50 µg CfDx dsRNA, the expression level of CfDx mRNA in haemocytes began to decrease significantly at 36 h and maintained the relative low level (about 0.3-fold of the PBS control group) from 36 to72 h, but the ratio of T4/T3 in haemolymph began to increase at 36 h (2.31-fold, P < 0.05) and kept increasing from 36 to 72 h comparing with that in the PBS control group. These results indicated that CfDx was a homologue of iodothyronine deiodinase in scallop C. farreri, and it might be involved in the immunomodulation via regulating the concentration of thyroid hormones T3 and T4 in the haemolymph of scallop.

The phenoloxidase activity and antibacterial function of a tyrosinase from scallop Chlamys farreri.

Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.

The Caspase Homologues in Scallop Chlamys farreri and Their Expression Responses to Toxic Dinoflagellates Exposure.

The cysteine aspartic acid-specific protease (caspase) family is distributed across vertebrates and invertebrates, and its members are involved in apoptosis and response to cellular stress. The Zhikong scallop (Chlamys farreri) is a bivalve mollusc that is well adapted to complex marine environments, yet the diversity of caspase homologues and their expression patterns in the Zhikong scallop remain largely unknown. Here, we identified 30 caspase homologues in the genome of the Zhikong scallop and analysed their expression dynamics during all developmental stages and following exposure to paralytic shellfish toxins (PSTs). The 30 caspase homologues were classified as initiators (caspases-2/9 and caspases-8/10) or executioners (caspases-3/6/7 and caspases-3/6/7-like) and displayed increased copy numbers compared to those in vertebrates. Almost all of the caspase-2/9 genes were highly expressed throughout all developmental stages from zygote to juvenile, and their expression in the digestive gland and kidney was slightly influenced by PSTs. The caspase-8/10 genes were highly expressed in the digestive gland and kidney, while PSTs inhibited their expression in these two organs. After exposure to different Alexandrium PST-producing algae (AM-1 and ACDH), the number of significantly up-regulated caspase homologues in the digestive gland increased with the toxicity level of PST derivatives, which might be due to the higher toxicity of GTXs produced by AM-1 compared to the N-sulphocarbamoyl analogues produced by ACDH. However, the effect of these two PST-producing algae strains on caspase expression in the kidney seemed to be stronger, possibly because the PST derivatives were transformed into highly toxic compounds in scallop kidney, and suggested an organ-dependent response to PSTs. These results indicate the dedicated control of caspase gene expression and highlight their contribution to PSTs in C. farreri. This work provides a further understanding of the role of caspase homologues in the Zhikong scallop and can guide future studies focussing on the role of caspases and their interactions with PSTs.

A dopamine beta hydroxylase from Chlamys farreri and its induced mRNA expression in the haemocytes after LPS stimulation.

Dopamine beta hydroxylase (DBH) is a critical enzyme in the biosynthesis of catecholamines, and also plays an important role in complex neuroendocrine-immune regulatory network. In the present study, the cDNA encoding dopamine beta hydroxylase (designated CfDBH) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The full-length cDNA of CfDBH was of 2302 bp, containing a 5' untranslated region (UTR) of 32 bp, a 3' UTR of 461 bp with a poly (A) tail, and an open reading frame (ORF) of 1809 bp encoding a polypeptide of 603 amino acids. The deduced amino acid sequence of CfDBH contained a signal peptide, a DOMON domain and a Cu2_monooxygen domain, and it shared 39.4%-42.9% similarity with other reported DBHs. The conserved domains in CfDBH and the amino acid sequence similarity with other DBHs strongly suggested that it was a homologue of DBH in C. farreri. The mRNA expression of CfDBH in various tissues and its temporal expression in haemocytes of scallops stimulated with LPS were ascertained by Quantitative real-time RT-PCR. The mRNA transcripts of CfDBH were detected in all the examined tissues with the highest expression level in hepatopancreas. The expression level of CfDBH in haemocytes was up-regulated after LPS stimulation and increased to hundreds fold higher than that of the control group at 12 h, and then decrease significantly to 0.36-fold and 0.31-fold at 24 h and 48 h respectively. The results suggested pathogen infections significantly induced the expression level of CfDBH, and the activation of DBH could influence the immune response of scallop C. farreri through changing the concentration of catecholamines.

Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α.

Phenylalanine hydroxylase (PAH) is an important metabolic enzyme of aromatic amino acids, which is responsible for the irreversible oxidation of phenylalanine to tyrosine. In the present study, the full-length cDNA encoding PAH from Chlamys farreri (designated CfPAH) was cloned by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfPAH encoded a polypeptide of 460 amino acids, and its sequence shared 64.4-74.2% similarity with those of PAHs from other animals. There were an N-terminal regulatory ACT domain and a C-terminal catalytic Biopterin_H domain in the deduced CfPAH protein. The mRNA transcripts of CfPAH could be detected in all the tested tissues, including adductor muscle, mantle, gill, gonad, haemocytes and hepatopancreas. And its expression level in haemocytes was increased significantly during 3-48 h after bacteria Vibrio anguillarum challenge with the highest level (9.1-fold, P < 0.05) at 24 h. Furthermore, the mRNA expression of CfPAH in haemocytes also increased significantly to 2.6-fold (P < 0.05) at 4 h and 3.3-fold (P < 0.05) at 6 h after the stimulation of 50.0 ng mL(-1) human TNF-α. The cDNA fragment encoding the mature peptide of CfPAH was recombined and expressed in the prokaryotic expression system, and 1 mg recombinant CfPAH protein (rCfPAH) could catalyze the conversion of 192.23 ± 32.35 nmol phenylalanine to tyrosine within 1 min (nmol min(-1) mg(-1) protein) in vitro. These results indicated collectively that CfPAH, as a homologue of phenylalanine hydroxylase in scallop C. farreri, could be induced by cytokine and involved in the immunomodulation of scallops by supplying the starting material tyrosine for the synthesis of melanin and catecholamines.

Identification and characterization of cellouronate (ß-1,4-linked polyglucuronic acid) lyase from the scallop Mizuhopecten yessoensis.

The semisynthetic polysaccharide cellouronate is a ß-1,4-linked polyglucuronic acid prepared from regenerated cellulose by chemical oxidation. Here, we isolated a novel enzyme, MyAly, as a cellouronate lyase from a scallop Mizuhopecten yessoensis. Its optimum temperature, pH, and NaCl concentration for cellouronate degradation were determined to be 30 °C, 6.9, and 200-500 mM, respectively. MyAly endolytically degraded cellouronate into unsaturated di-, tri-, and tetrasaccharides with kcat of 31.1 s-1. MyAly also showed an alginate-degradation activity with a kcat value of 0.58 s-1. However, there was no significant difference in Km values between cellouronate and alginate. MyAly consisted of 280 amino acids and shared 36.5-44.1 % identity with known marine gastropod alginate lyases belonging to the polysaccharide lyase family 14. This is the first study to identify and characterize a cellouronate-degrading lyase from a marine organism, providing a better understanding of the biodegradability of the industrially important polysaccharide, cellouronate, in marine environments.