Molluscan pleiotropic FADD involved in innate immune signaling and induces apoptosis.

Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon ß and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.

Insights into ADAR gene complement, expression patterns, and RNA editing landscape in Chlamys farreri.

Adenosine Deaminases Acting on RNA (ADARs) are evolutionarily conserved enzymes known to convert adenosine to inosine in double-stranded RNAs and participate in host-virus interactions. Conducting a meta-analysis of available transcriptome data, we identified and characterised eight ADAR transcripts in Chlamys farreri, a farmed marine scallop susceptible to Acute viral necrosis virus (AVNV) infections and mortality outbreaks. Accordingly, we identified six ADAR genes in the Zhikong scallop genome, revised previous gene annotations, and traced alternative splicing variants. In detail, each ADAR gene encodes a unique combination of functional domains, always including the Adenosine deaminase domain, RNA binding domains and, in one case, two copies of a Z-DNA binding domain. After phylogenetic analysis, five C. farreri ADARs clustered in the ADAR1 clade along with sequences from diverse animal phyla. Gene expression analysis indicated CF051320 as the most expressed ADAR, especially in the eye and male gonad. The other four ADAR1 genes and one ADAR2 gene exhibited variable expression levels, with CF105370 and CF051320 significantly increasing during early scallop development. ADAR-mediated single-base editing, evaluated across adult C. farreri tissues and developmental stages, was mainly detectable in intergenic regions (83 % and 85 %, respectively). Overall, the expression patterns of the six ADAR genes together with the editing and hyper-editing values computed on scallops RNA-seq samples support the adaptive value of ADAR1-mediated editing, particularly in the pre-settling larval stages.

The involvement of CaMKKI in activating AMPKα in yesso scallop Patinopecten yessoensis under high temperature stress.

Calcium/calmodulin dependent protein kinase kinase (CaMKK), a highly conserved protein kinase, is involved in the downstream processes of various biological activities by phosphorylating and activating 5'-AMP-activated protein kinase (AMPK) in response to the increase of cytosolic-free calcium (Ca2+). In the present study, a CaMKKI was identified from Yesso scallop Patinopecten yessoensis. Its mRNA was ubiquitously expressed in haemocytes and all tested tissues with the highest expression level in mantle. The expression level of PyCaMKKI mRNA in adductor muscle was significantly upregulated at 1, 3 and 6 h after high temperature treatment (25 °C), which was 3.43-fold (p < 0.05), 5.25-fold (p < 0.05), and 5.70-fold (p < 0.05) of that in blank group, respectively. At 3 h after high temperature treatment (25 °C), the protein level of PyAMPKα, as well as the phosphorylation level of PyAMPKα at Thr170 in adductor muscle, and the positive co-localized fluorescence signals of PyCaMKKI and PyAMPKα in haemocyte all increased significantly (p < 0.05) compared to blank group (18 °C). The pull-down assay showed that rPyCaMKKI and rPyAMPKα could bind each other in vitro. After PyCaMKKI was silenced by siRNA, the mRNA and protein levels of PyCaMKKI and PyAMPKα, and the phosphorylation level of PyAMPKα at Thr170 in adductor muscle were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. These results collectively suggested that PyCaMKKI was involved in the activation of PyAMPKα in response to high temperature stress and would be helpful for understanding the function of PyCaMKKI-PyAMPKα pathway in maintaining energy homeostasis under high temperature stress in scallops.

Coordination regulation of enhanced performance reveals the tolerance mechanism of Chlamys farreri to azaspiracid toxicity.

Azaspiracids (AZAs) are lipid biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. However, the mechanisms underlying the tolerance of shellfish to high levels of such toxins remain poorly understood. This study investigated the combined effects of detoxification metabolism and stress-related responses in scallops Chlamys farreri exposed to AZA. Scallops accumulated a maximum of 361.81 µg AZA1 eq/kg and 41.6 % AZA residue remained after 21 days of exposure. A range of AZA2 metabolites, including AZA19, AZA11, and AZA23, and trace levels of AZA2-GST, were detected. Total hemocyte counts significantly increased and ROS levels remained consistently high until gradually decreasing. Immune system activation mediated mitochondrial dysfunction and severe energy deficiency. DEGs increased over time, with key genes CYP2J6 and GPX6 contributing to AZA metabolism. These transcriptome and metabolic results identify the regulation of energy metabolism pathways, including inhibition of the TCA cycle and activation of carbohydrates, amino acids, and lipids. AZA also induced autophagy through the MAPK-AMPK signaling pathways, and primary inhibited PI3K/AKT to decrease mTOR pathway expression. Our results provide additional insights into the resistance of C. farreri to AZA, characterized by re-establishing redox homeostasis toward a more oxidative state.

A newly identified scallop MyD88 interacts with TLR and functions in innate immunity.

Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-ß, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future.

Effects of microbial community and disease resistance against Vibrio splendidus of Yesso scallop (Patinopecten yessoensis) fed supplementary diets of tussah immunoreactive substances and antimicrobial peptides.

This study was conducted to investigate the effects of dietary supplementation of tussah immunoreactive substances (TIS) and antimicrobial peptides (AMPs) on microbial community and resistance against Vibrio splendidus of Yesso scallop Patinopecten yessoensis. Scallops were fed with the basal diets supplemented with TIS (T group), AMPs (A group), or both of the two (TA group). After the feeding trial, the microbial community changes were evaluated, and the challenge test with V. splendidus was conducted, as well as the immune parameters and digestive enzyme activities were determined. The results revealed that the TA group was more capable of modulating the bacterial community composition of scallops by increasing the potentially beneficial bacteria and suppressing the pathogenic microorganism during the feeding trial. After injection, the cumulative mortality rate in TA group was notably lower than others. In addition, the TA group showed better digestive and immune parameters involved in digestive capacity, phagocyte function, phosphatase-responsiveness, and oxidation resistance. These results collectively confirmed that dietary TIS and AMPs in diet could effectively modulate the microflora structure and improve disease resistance against V. splendidus of scallop, and the positive effects were more obvious when dietary supplementation of them in combination.

The involvement of PyBeclin 1 and PyLC3 in regulating the activation of autophagy in scallop Patinopecten yessoensis after acute high temperature stress.

Beclin 1 and LC3 are important autophagy regulation proteins involved in vesicle nucleation and extension stage, respectively. In the present study, a Beclin 1 and a LC3 were identified from Yesso scallop Patinopecten yessoensis (PyBeclin 1 and PyLC3). The open reading frame (ORF) of PyBeclin 1 was of 1335 bp encoding a putative polypeptide of 444 amino acid residues with an N-terminal BCL-2 homology 3 (BH3) domain, a central coiled-coil domain (CCD), and a C-terminal evolutionarily conserved domain (ECD). The ORF of PyLC3 was of 369 bp encoding a putative polypeptide of 122 amino acid residues with an APG12 domain. The deduced amino acid sequences of PyBeclin 1 and PyLC3 shared 31.92-74.09% and 68.38-79.50% identities with Beclin 1s and LC3s from other species, respectively. The mRNA transcripts of PyBeclin 1 and PyLC3 were found to be expressed in all the examined tissues, including adductor muscle, gonad, gill, haemocytes and mantle, with the highest expression level in gill and haemocytes. The mRNA expression level of PyBeclin 1 in haemocytes increased significantly at 1, 3, 6, 12 and 24 h (2.98-4.07 fold of that in the Blank group, p < 0.05), and returned to normal level at 48 h after acute high temperature stress at 25 °C. Unlike PyBeclin 1, the mRNA transcripts of PyLC3 in haemocytes were significantly up-regulated at1, 3, 6 and 12 h (1.80-2.54 fold of that in the Blank group, p < 0.05), then decreased to blank level at 24 h (p > 0.05), and increased significantly again at 48 h (3.70 fold of that in the Blank group, p < 0.05) after high temperature. PyBeclin 1 and PyLC3 were mainly located in the cytoplasm and a small amount in the nucleus with few puncta, and the numbers of PyBeclin 1 and PyLC3 puncta increased at 3 h after acute high temperature stress. The LC3-II levels in gill and haemocytes were significantly up-regulated at 1 h and 3 h after acute high temperature stress. These results collectively suggested that PyBeclin 1 and PyLC3 were conserved members of Beclin 1 and LC3 family in scallops, and involved in regulating the activation of autophagy in scallops after acute high temperature stress.

Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph.

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.

NKL-24: A novel antimicrobial peptide derived from zebrafish NK-lysin that inhibits bacterial growth and enhances resistance against Vibrio parahaemolyticus infection in Yesso scallop, Patinopecten yessoensis.

The extensive use of antibiotics in aquaculture has resulted in the prevalence of antibiotic-resistant bacteria and, consequently, new antibacterial strategies or drugs with clear modes of action are urgently needed. Antimicrobial peptides (AMPs) are currently widely considered as alternatives to antibiotics in the treatment of infections in aquatic animals. In this study, we aimed to evaluate the effects of NKL-24, a truncated peptide derived from zebrafish NK-lysin, against Yesso scallop (Patinopecten yessoensis) pathogen, Vibrio parahaemolyticus. The results showed that NKL-24 had a potent antibacterial effect against V. parahaemolyticus via a membrane active cell-killing mechanism. The in vitro study showed that sub-lethal levels of NKL-24 obviously reduced bacterial swimming ability and downregulated the transcription of the selected genes associated with V. parahaemolyticus virulence. Studies on NKL-24 biosafety in hemocytes and in Yesso scallop have shown no adverse effects from this peptide. Bacteria challenge test results demonstrated that NKL-24 significantly decreased the mortality and inhibited bacterial growth in the scallop infected with V. parahaemolyticus, while further in vivo examination revealed that NKL-24 could enhance non-specific immune parameters. Moreover, NKL-24 was capable of modulating a series of V. parahaemolyticus-responsive genes in the scallop. These results suggest the protective action of NKL-24 against V. parahaemolyticus and the potential of this peptide as a promising candidate for aquaculture applications.

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.

Effects of high stocking density on the galectin gene expression in noble scallop Chalmys nobilis under bacterial infection.

High stocking densities have been shown to have adverse effects on the physiology of bivalves. The noble scallop Chlamys nobilis is one of the most important cultured shellfish in Southern China. However, the effects of scallop stocking density on its immunity is not well understood. In this context, this study was conducted to assess the effect of high stocking density on the galectin (an important protein in innate immunity) gene expression of C. nobilis during bacterial infection. A full-length galectin (CnGal) gene was cloned. The ORF of the CnGal cDNA encodes a predicted protein containing 549 aa with four CRDs and no signal peptide. Our results reveal that high stocking density in the scallop not only led to high mortality and slow growth, but also changed tissue distribution of the CnGal expression. The individuals from the high stocking density group exhibited more differences among tissues than those from the control group, but the highest expression were both recorded in hemolymph. After the Vibrio parahaeomlyticus challenge, the gene's expression levels were all significantly up-regulated in the hemolymph and gill, but the time up to peak was different between the two tissues. The findings of this study could fill a gap in knowledge about how high stocking density affect scallop immunity at the molecular level.

Transcriptome analysis of Catarina scallop (Argopecten ventricosus) juveniles treated with highly-diluted immunomodulatory compounds reveals activation of non-self-recognition system.

Marine bivalve hatchery productivity is continuously challenged by apparition and propagation of new diseases, mainly those related to vibriosis. Disinfectants and antibiotics are frequently overused to prevent pathogen presence, generating a potential negative impact on the environment. Recently, the use of highly diluted compounds with immunostimulant properties in marine organisms has been trailed successfully to activate the self-protection mechanisms of marine bivalves. Despite their potential as immunostimulants, little is known about their way of action. To understand their effect, a comparative transcriptomic analysis was performed with Argopecten ventricosus juveniles. The experimental design consisted of four treatments formulated from pathogenic Vibrio lysates at two dilutions: [(T1) Vibrio parahaemolyticus and Vibrio alginolyticus 1D; (T2) V. parahaemolyticus and V. alginolyticus 7C]; minerals [(T3) PhA+SiT 7C], scorpion venom [(T4) ViT 31C]; and one control (C1) hydro-alcoholic solution (ethanol 1%). The RNA sequencing (RNAseq) analysis showed a higher modulation of differentially expressed genes (DEG) in mantle tissue compared to gill tissue. The scallops that showed a higher number of DEG related to immune response in mantle tissue corresponded to T1 (V. parahaemolyticus and V. alginolyticus lysate) and T3 (Silicea terra® - Phosphoric acid®). The transcriptome analysis allowed understanding some interactions between A. ventricosus juveniles and highly-diluted treatments.

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.

Identification and molecular characterization of peroxiredoxin 6 from noble scallop Chlamys nobilis revealing its potent immune response and antioxidant property.

The 1-cyseine peroxiredoxin (Prx6) is an importantly antioxidant enzyme that protects cells from oxidative damage caused by excessive production of reactive oxygen species (ROS). In this study, we described the molecular characteristics of the noble scallop Chlamys nobilis peroxiredoxin 6 (designed as CnPrx6), immune responses and DNA protection activity of the recombinant protein. The complete ORF (696 bp) of CnPrx6 encoded a polypeptide (25.5 kDa) of 231 amino acids, harboring a conserved peroxidase catalytic center (41PVCTTE46) and the catalytic triads putatively involved in peroxidase and phospholipase A2 activities. The deduced amino acid sequence of CnPrx6 shared a relatively high amino acid sequence similarity (more than 50%). The qRT-PCR revealed that the CnPrx6 mRNA was constitutively expressed in all examined tissues, with the highest expression observed in adductor. Upon immunological challenge with Vibrio parahaemolyticus, lipopolysaccharides (LPS) and polyinosinic-polycytidylic acid (Poly I:C), the expression level of CnPrx6 mRNA was significantly up-regulated (P < 0.05). Furthermore, there was a significant difference (P < 0.05) in the expression level of CnPrx6 between golden and brown scallops. The purified recombinant CnPrx6 protein protected the supercoiled plasmid DNA from metal-catalyzed ROS damage. Taken together, these results indicated that the CnPrx6 may play an important role in modulating immune responses and minimizing DNA damage in noble scallop Chlamys nobilis.

Molecular characterization and expression patterns of two LPS binding /bactericidal permeability-increasing proteins (LBP/BPIs) from the scallop Argopecten purpuratus.

Lipopolysaccharide-binding proteins (LBPs) and bactericidal permeability-increasing proteins (BPIs) are effectors of the innate immune response which act in a coordinated manner to bind and neutralize the LPS present in Gram negative bacteria. The structural organization that confers the function of LBPs and BPIs is very similar, however, they are antagonistic to each other. In this work, we characterized two LBP/BPIs from the scallop Argopecten purpuratus, namely ApLBP/BPI1 and ApLBP/BPI2. The molecular and phylogenetic analyses of ApLBP/BPIs indicated that both isoforms display classic characteristics of LBP/BPIs from other invertebrates. Additionally, ApLBP/BPIs are constitutively expressed in scallop tissues and their transcript expression is upregulated in hemocytes and gills in response to an immune challenge. However, some structural characteristics of functional importance for the biological activity of these molecules, such as the net charge differ substantially between ApLBP/BPI1 and ApLBP/BPI2. Furthermore, each isoform displays a specific profile of basal expression among different tissues, as well as specific patterns of expression during the activation of the immune response. Results suggest that functional specialization of ApLBP/BPIs might happen, with potential role as LBP or BPI in this species of scallop. Further research on the biological activities of ApLBP/BPIs are necessary to elucidate their participation in the scallop immune response.

Effects of thermal stress on mortality and HSP90 expression levels in the noble scallops Chlamys nobilis with different total carotenoid content.

The noble scallop Chlamys nobilis is an economically important marine bivalve cultivated in the southern sea of China since the 1980s. Unfortunately, mass mortality of this scallop species often occurs in summer. The present study was conducted to investigate whether the expression of heat shock protein 90 (HSP90) and level of carotenoids could enhance high-temperature stress resistance in scallop. First, the HSP90 homolog of C. nobilis (designated CnHSP90) was identified and cloned. The complete cDNA sequence of CnHSP90 was 2631 bp, including a 2181-bp open reading frame (ORF) encoding a 726 amino acid polypeptide with five HSP90 family signatures, and sharing high homology with members of the HSP90 family. CnHSP90 was ubiquitously expressed in all examined tissues including the intestine, kidney, adductor, mantle, gill, and gonad, with the highest in the gonad. Golden and brown scallops, which contain significantly different total carotenoid content (TCC), were subjected to acute thermal challenge, and the LTE50 (semi-lethal temperature at 36 h heat shock) and LTI50 (semi-lethal time after heat shock) as well as the correlation between CnHSP90 gene expression and TCC were determined. The LTE50 of golden scallop (32.14 °C) was higher than that of brown scallops (31.19 °C), with longer LTI50 at all tested temperatures, indicating that golden scallops were more resistant to thermal stress than brown scallops. Similarly, the mRNA expression levels of CnHSP90 in gill of golden scallops were significantly higher (P < 0.05) than that of brown scallops at 6, 12, 24, and 36 h, with a strong positive correlation between CnHSP90 expression level and TCC. This suggests that both carotenoids and HSP90 levels could improve thermal resistance in the noble scallops.

Oxidative stress responses of golden and brown noble scallops Chlamys nobilis to acute cold stress.

The noble scallop Chlamys nobilis is an important edible marine bivalve that is widely cultivated in the sea of southern China. Unfortunately, the mass mortality of noble scallops frequently occurs during the winter months. The present study investigated the effects of acute cold stress (8 °C) to the physiological responses of polymorphic noble scallops, by assessing the HSP70 gene expression, total carotenoid content (TCC), total antioxidant capacity (TAC), malondialdehyde (MDA) content, catalase (CAT) activity and superoxide dismutase (SOD) enzymatic activity in different tissues of golden and brown scallops. The results of the present study revealed that MDA, TCC and CAT increased drastically in most tissues in the early stage of acute cold stress (0-3 h), but TCC, SOD and CAT generally showed a downward trend. Within 3-6 h of acute cold stress, MDA content decreased in most tissues and the SOD content increased significantly in most tissues, while TCC and CAT remained at peak. After 6 h of acute cold stress, MDA content continued to increase in most tissues, while TCC, CAT, SOD and TAC decreased or remained at a lower level. For HSP70 expression, up-regulation of the HSP70 gene was observed only in mantle of brown scallops and hemolymph of golden scallops at 3 h and 24 h, respectively. The findings of the present study can better understand the physiological response of noble scallops to acute cold stress.

Alternative splicing, spatiotemporal expression of TEP family genes in Yesso scallop (Patinopecten yessoensis) and their disparity in responses to ocean acidification.

The complement system constitutes a highly sophisticated and powerful body defense machinery acting in the innate immunity of both vertebrates and invertebrates. As central components of the complement system, significant effects of thioester-containing protein (TEP) family members on immunity have been reported in most vertebrates and in some invertebrates, but the spatiotemporal expression and regulatory patterns of TEP family genes under environmental stress have been less widely investigated in scallops. In this study, expression profiling of TEP family members in the Yesso scallop Patinopecten yessoensis (designated PyTEPs) was performed at all developmental stages, in different healthy adult tissues, and in mantles during exposure to different levels of acidification (pH = 6.5 and 7.5) for different time points (3, 6, 12 and 24 h); this profiling was accomplished through in silico analysis of transcriptome and genome databases. Spatiotemporal expression patterns revealed that PyTEPs had specific functional differentiation in all stages of growth and development of the scallop. Expression analysis confirmed the inducible expression patterns of PyTEPs during exposure to acidification. Gene duplication and alternative splicing events simultaneously occurred in PyTEP1. Seven different cDNA variants of PyTEP1 (designated PyTEP1-A-PyTEP1-G) were identified in the scallop mantle transcriptome during acidic stress. These variants were produced by the alternative splicing of seven differentially transcribed exons (exons 18-24), which encode the highly variable central region. The responses to immune stress may have arisen through the gene duplication and alternative splicing of PyTEP1. The sequence diversity of PyTEP1 isoforms and their different expression profiles in response to ocean acidification (OA) suggested a mechanism used by scallops to differentiate and regulate PyTEP1 gene expression. Collectively, these results demonstrate the gene duplication and alternative splicing of TEP family genes and provide valuable resources for elucidating their versatile roles in bivalve innate immune responses to OA challenge.

Differential expressions of HSP70 gene between golden and brown noble scallops Chlamys nobilis under heat stress and bacterial challenge.

Heat shock proteins (HSPs) are a family of conserved proteins that enhance stress resistance and protect cells from external damage. In the present study, the full-length HSP70 cDNA from the noble scallop Chlamys nobilis (designated CnHSP70) was first cloned and characterized. Then, the expression of CnHSP70 in golden and brown scallops with different carotenoid content was evaluated under heat stress and Vibrio parahaemolyticus challenge. The complete CnHSP70 cDNA is 2621 bp, including a 1971 bp open reading frame (ORF) encoding a polypeptide of 656 amino acids with an estimated molecular weight of 71.55 kDa and an isoelectric point of 5.32. Based on amino acid sequence and phylogenetic analysis, the CnHSP70 gene was identified as a member of the cytoplasmic HSP70 family. The CnHSP70 was ubiquitously expressed in all examined tissues, including intestines, hemocytes, mantle, adductor and gills, with the highest expression in gills. After heat stress and V. parahaemolyticus injection, the expression levels of CnHSP70 in gills and hemocytes of golden and brown scallops were both significantly increased, indicating that the gene was involved in resistance or immune response. Moreover, under both conditions, similar expression profiles of CnHSP70 were observed between gills and hemocytes from the same color scallop, but different expression levels were detected in the same tissue from the different color scallop, which may be related to difference in their carotenoids content.

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.