Novel microbial fermentation for the preparation of iron-chelating scallop skirts peptides-its profile, identification, and possible binding mode.

Iron chelating peptides have been widely utilized as iron supplements due to their excellent absorption capacity, However, the high cost and cumbersome manufacturing process of these peptides significantly limit their industrial application. In this study, fermentation was used for the first time to prepare iron chelating peptides. Bacillus altitudinis 3*1-3 was selected as the most suitable strain from 50 strains. The hydrolysates of fermented scallop skirts showed excellent iron-chelating capacity (9.39 mg/g). Aspartic acid, glutamic acid, and histidine are crucial for the binding of peptides to ferrous ions. The heptapeptide (FEDPEFE) forms six binding bonds with ferrous irons. Compared with ferrous sulfate, peptide-ferrous chelate showed more stability in salt solution and simulated gastrointestinal juice (p < 0.05). Furthermore, the fermentation method could save >50% of the cost compared with the enzymatic method. The results can provide a theoretical basis for the preparation of ferrous-chelated peptides using the fermentation method.

Somatostatin Receptor Gene Functions in Growth Regulation in Bivalve Scallop and Clam.

Bivalves hold an important role in marine aquaculture and the identification of growth-related genes in bivalves could contribute to a better understanding of the mechanism governing their growth, which may benefit high-yielding bivalve breeding. Somatostatin receptor (SSTR) is a conserved negative regulator of growth in vertebrates. Although SSTR genes have been identified in invertebrates, their involvement in growth regulation remains unclear. Here, we identified seven SSTRs (PySSTRs) in the Yesso scallop, Patinopecten yessoensis, which is an economically important bivalve cultured in East Asia. Among the three PySSTRs (PySSTR-1, -2, and -3) expressed in adult tissues, PySSTR-1 showed significantly lower expression in fast-growing scallops than in slow-growing scallops. Then, the function of this gene in growth regulation was evaluated in dwarf surf clams (Mulinia lateralis), a potential model bivalve cultured in the lab, via RNA interference (RNAi) through feeding the clams Escherichia coli containing plasmids expressing double-stranded RNAs (dsRNAs) targeting MlSSTR-1. Suppressing the expression of MlSSTR-1, the homolog of PySSTR-1 in M. lateralis, resulted in a significant increase in shell length, shell width, shell height, soft tissue weight, and muscle weight by 20%, 22%, 20%, 79%, and 92%, respectively. A transcriptome analysis indicated that the up-regulated genes after MlSSTR-1 expression inhibition were significantly enriched in the fat digestion and absorption pathway and the insulin pathway. In summary, we systemically identified the SSTR genes in P. yessoensis and revealed the growth-inhibitory role of SSTR-1 in bivalves. This study indicates the conserved function of somatostatin signaling in growth regulation, and ingesting dsRNA-expressing bacteria is a useful way to verify gene function in bivalves. SSTR-1 is a candidate target for gene editing in bivalves to promote growth and could be used in the breeding of fast-growing bivalves.

The Glucose-Succinate Pathway: A Crucial Anaerobic Metabolic Pathway in the Scallop Chlamys farreri Experiencing Heat Stress.

Recently, the increase in marine temperatures has become an important global marine environmental issue. The ability of energy supply in marine animals plays a crucial role in avoiding the stress of elevated temperatures. The investigation into anaerobic metabolism, an essential mechanism for regulating energy provision under heat stress, is limited in mollusks. In this study, key enzymes of four anaerobic metabolic pathways were identified in the genome of scallop Chlamys farreri, respectively including five opine dehydrogenases (CfOpDHs), two aspartate aminotransferases (CfASTs) divided into cytoplasmic (CfAST1) and mitochondrial subtype (CfAST2), and two phosphoenolpyruvate carboxykinases (CfPEPCKs) divided into a primitive type (CfPEPCK2) and a cytoplasmic subtype (CfPEPCK1). It was surprising that lactate dehydrogenase (LDH), a key enzyme in the anaerobic metabolism of the glucose-lactate pathway in vertebrates, was absent in the genome of scallops. Phylogenetic analysis verified that CfOpDHs clustered according to the phylogenetic relationships of the organisms rather than substrate specificity. Furthermore, CfOpDHs, CfASTs, and CfPEPCKs displayed distinct expression patterns throughout the developmental process and showed a prominent expression in muscle, foot, kidney, male gonad, and ganglia tissues. Notably, CfASTs displayed the highest level of expression among these genes during the developmental process and in adult tissues. Under heat stress, the expression of CfASTs exhibited a general downregulation trend in the six tissues examined. The expression of CfOpDHs also displayed a downregulation trend in most tissues, except CfOpDH1/3 in striated muscle showing significant up-regulation at some time points. Remarkably, CfPEPCK1 was significantly upregulated in all six tested tissues at almost all time points. Therefore, we speculated that the glucose-succinate pathway, catalyzed by CfPEPCK1, serves as the primary anaerobic metabolic pathway in mollusks experiencing heat stress, with CfOpDH3 catalyzing the glucose-opine pathway in striated muscle as supplementary. Additionally, the high and stable expression level of CfASTs is crucial for the maintenance of the essential functions of aspartate aminotransferase (AST). This study provides a comprehensive and systematic analysis of the key enzymes involved in anaerobic metabolism pathways, which holds significant importance in understanding the mechanism of energy supply in mollusks.

Potential roles of hydroxybenzoate paralytic shellfish toxins in modulating toxin biokinetics in scallops.

Paralytic shellfish toxins (PSTs) produced by some marine dinoflagellates can cause severe human intoxication via vectors like bivalves. Toxic dinoflagellate Gymnodinium catenatum produce a novel group of hydroxybenzoate PSTs named GC toxins, but their biokinetics in bivalves haven't been well examined. In this experiment, we analyzed PSTs in bay scallops Argopecten irradians exposed to G. catenatum (strain MEL11) to determine their accumulation, elimination, anatomical distribution, and biotransformation. To our surprise, up to 30% of the PSTs were accumulated in the adductor muscle of scallops at the end of the experiment, and the toxicity of adductor muscle exceeded the regulatory limit of 800 µg STXeq/kg in only 6 days. High concentration of toxins in the adductor muscle are likely linked to the rapid transfer of GC toxins from viscera to other tissues. Moreover, most GC toxins in scallops were found rapidly transformed to decarbamoyl toxins through enzyme-mediated hydrolysis, which was further supported by the in vitro incubation experiments. Our study demonstrates that GC toxins actively participate in toxin distribution and transformation in scallops, which may increase the risks of food poisoning associated with the consumption of scallop adductor muscle. ENVIRONMENTAL IMPLICATION: The negative impacts of harmful algal blooms (HABs) have become a global environmental concern under the joint effects of cultural eutrophication and climate change. Our study, targeted on the biokinetics of paralytic shellfish toxins in scallops exposed to Gymnodinium catenatum producing unique GC toxins, aims to elucidate potential risks of seafood poisoning associated with GC toxins. The findings of this study will help us to understand the roles of GC toxins in seafood poisoning, and to develop effective management strategies against toxic algal blooms and phycotoxins.

Effects of chronic cold stress on tissue structure, antioxidant response, and key gene expression in the warm-water bivalve Chlamys nobilis.

As ectothermic invertebrates, mollusks are regarded as good environmental indicator species for determining the adverse effects of climate change on marine organisms. In the present study, the effects of cold stress on the tissue structure, antioxidant activity, and expression levels of genes were evaluated in the warm-water noble scallop Chlamys nobilis by simulating natural seawater cooled down during winter from 17 °C to 14 °C, 12 °C, 10 °C, and 9 °C. Firstly, the gill was severely damaged at 10 °C and 9 °C, indicating that it could be used as a visually indicative organ for monitoring cold stress. The methylenedioxyamphetamine (MDA) content significantly increased with the temperatures decreasing, meanwhile, the antioxidant enzyme activities superoxide dismutase (SOD) and catalase (CAT) showed a similar pattern, suggesting that the scallop made a positive response. More importantly, 6179 genes related to low temperatures were constructed in a module-gene clustering heat map including 10 modules. Furthermore, three gene modules about membrane lipid metabolism, amino acid metabolism, and molecular defense were identified. Finally, six key genes were verified, and HEATR1, HSP70B2, PI3K, and ATP6V1B were significantly upregulated, while WNT6 and SHMT were significantly downregulated under cold stress. This study provides a dynamic demonstration of the major gene pathways' response to various low-temperature stresses from a transcriptomic perspective. The findings shed light on how warm-water bivalves can tolerate cold stress and can help in breeding new strains of aquatic organisms with low-temperature resistance.

Effect of acute cadmium exposure on oxidative stress and antioxidant system of the scallop Aequipecten tehuelchus.

This study aimed to assess the impact of acute exposure (96 h) to Cd in gills, digestive gland and muscle of the Tehuelche scallop Aequipecten tehuelchus from San José gulf in Patagonia, Argentina. Scallops were exposed to Cd concentrations of 0, 25, 50, 100, 150, 204, 275, 371, and 500 µg/L, and mortality rates were recorded after 96 h of exposure. Surviving organisms were analyzed for the biochemical response through reactive oxygen and nitrogen species (RONS), activities of catalase (CAT) and glutathione-S-transferase (GST), metallothioneins (MT), lipid peroxidation (LPO) and liposoluble antioxidants α-tocopherol (α-T) and ß-carotene (ß-C). The mean lethal concentration (LC50) was 155.8 µg Cd/L, a lower value than other scallops' species, showing that A. tehuelchus has a particular sensitivity to Cd. In the three tissues, at all exposure concentrations, there was no significant response in RONS levels, GST activity or LPO. Nevertheless, CAT activity and α-T levels decreased in the gills but increased in the digestive gland, with no significant response in the muscle. Two-way ANOVA revealed a significant interaction between Cd concentration and tissue on MT, which increased significantly in gills, decreased in digestive gland with 100 compared to 50 µg Cd/L; whereas in muscle a significant increase was observed with 25 µg Cd/L compared to control. The results show a significant effect of Cd in scallop's gills on CAT activity and α-T levels, highlighting this tissue as the primary target against relevant concentrations of metal in seawater. The effect on digestive gland and muscle was minimal. The overall results suggest that Cd toxicity is tissue-specific. This study will help reduce the existence knowledge gap regarding potential impacts of acute exposure to Cd in a bivalve species with high ecological and commercial importance, as well as identifying the most responsive biomarkers associated with Cd stress for monitoring assessment.

Genome-wide investigation of the TGF-ß superfamily in scallops.

BACKGROUND: Transforming growth factor ß (TGF-ß) superfamily genes can regulate various processes, especially in embryogenesis, adult development, and homeostasis. To understand the evolution and divergence patterns of the TGF-ß superfamily in scallops, genome-wide data from the Bay scallop (Argopecten irradians), the Zhikong scallop (Chlamys farreri) and the Yesso scallop (Mizuhopecten yessoensis) were systematically analysed using bioinformatics methods. RESULTS: Twelve members of the TGF-ß superfamily were identified for each scallop. The phylogenetic tree showed that these genes were grouped into 11 clusters, including BMPs, ADMP, NODAL, GDF, activin/inhibin and AMH. The number of exons and the conserved motif showed some differences between different clusters, while genes in the same cluster exhibited high similarity. Selective pressure analysis revealed that the TGF-ß superfamily in scallops was evolutionarily conserved. The spatiotemporal expression profiles suggested that different TGF-ß members have distinct functions. Several BMP-like and NODAL-like genes were highly expressed in early developmental stages, patterning the embryonic body plan. GDF8/11-like genes showed high expression in striated muscle and smooth muscle, suggesting that these genes may play a critical role in regulating muscle growth. Further analysis revealed a possible duplication of AMH, which played a key role in gonadal growth/maturation in scallops. In addition, this study found that several genes were involved in heat and hypoxia stress in scallops, providing new insights into the function of the TGF-ß superfamily. CONCLUSION: Characteristics of the TGF-ß superfamily in scallops were identified, including sequence structure, phylogenetic relationships, and selection pressure. The expression profiles of these genes in different tissues, at different developmental stages and under different stresses were investigated. Generally, the current study lays a foundation for further study of their pleiotropic biological functions in scallops.

The immunotoxicity mechanism of hemocytes in Chlamys farreri incubated with noradrenaline and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide alone or in combination.

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is the active intermediate metabolite of benzo[a]pyrene (B[a]P) and is considered the ultimate immunotoxicant. The neuroendocrine immunoregulatory network of bivalves is affected under pollutant stress. Besides, bivalves are frequently affected by pollutants in marine environments, yet the combined effects of neuroendocrine factors and detoxification metabolites on bivalves under pollutant stress and the signal pathways that mediate this immunoregulation are not well understood. Therefore, we incubated the hemocytes of Chlamys farreri with the neuroendocrine factor noradrenaline (NA) and the B[a]P detoxification metabolite BPDE, alone or in combination, to examine the immunotoxic effects of NA and BPDE on the hemocytes in C. farreri. Furthermore, the effects of NA and BPDE on the hemocyte signal transduction pathway were investigated by assessing potential downstream targets. The results revealed that NA and BPDE, alone or in combination, resulted in a significant decrease in phagocytic activity, bacteriolytic activity and the total hemocyte count. In addition, the immunotoxicity induced by BPDE was further exacerbated by co-treatment with NA, and the two showed synergistic effects. Analysis of signaling pathway factors showed that NA activated G proteins by binding to α-AR, which transmitted information to the Ca2+-NF-κB signaling pathway to regulate the expression of phagocytosis-associated proteins and regulated cytokinesis through the cAMP signaling pathway. BPDE could activate PTK and affect phagocytosis and cytotoxicity proteins through Ca2+-NF-κB signal pathway, also affect the regulation of phagocytosis and cytotoxicity by inhibiting the AC-cAMP-PKA pathway to down-regulate the expression of NF-κB and CREB. In addition, BPDE and NA may affect the immunity of hemocytes by down-regulating phagocytosis-related proteins through inhibition of the lectin pathway, while regulating the expression of cytotoxicity-related proteins through the C-type lectin. In summary, immune parameters were suppressed through Ca2+ and cAMP dependent pathways exposed to BPDE and the immunosuppressive effects were enhanced by the neuroendocrine factor NA.

Intrinsic and extrinsic pathways of apoptosis induced by multiple antibiotics residues and ocean acidification in hemocytes of scallop Argopecten irradians irradians: An interactionist perspective.

The increasing prevalence of antibiotics in seawater across global coastal areas, coupled with the ocean acidification induced by climate change, present a multifaceted challenge to marine ecosystems, particularly impacting the key physiological processes of marine organisms. Apoptosis is a critical adaptive response essential for maintaining cellular homeostasis and defending against environmental threats. In this study, bay scallops Argopecten irradians irradians were exposed to multiple antibiotics (sulfamethoxazole, tetracycline, oxytetracycline, norfloxacin, and erythromycin, each at a concentration of 1 µg/L) combined with/without acidic seawater (pH 7.6) for 35 days. The single and interactive effects of the two stressors on apoptosis and the underlying mechanisms in hemocytes of A. irradians irradians were determined through flow cytometry analysis, comet assay, oxidative stress biomarkers analysis, and transcriptome analysis. Results showed that apoptosis could be triggered by either AM exposure or OA exposure, but through different pathways. Exposure to AM leads to mitochondrial dysfunction and oxidative damage, which in turn triggers apoptosis via a series of cellular events in both intrinsic and extrinsic pathways. Conversely, while OA exposure similarly induced apoptosis, its effects are comparatively subdued and are predominantly mediated through the intrinsic pathway. Additionally, the synergistic effects of AM and OA exposure induced pronounced mitochondrial dysfunction and oxidative damages in the hemocytes of A. irradians irradians. Despite the evident cellular distress and the potential initiation of apoptotic pathways, the actual execution of apoptosis appears to be restrained, which might be attributed to an energy deficit within the hemocytes. Our findings underscore the constrained tolerance capacity of A. irradians irradians when faced with multiple environmental stressors, and shed light on the ecotoxicity of antibiotic pollution in the ocean under prospective climate change scenarios.

Effects of Benzo[a]pyrene on Food Metabolism and Reproductive Endocrine and Ovarian Development in Female Scallop Chlamys farreri at Different Reproductive Stages.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH) with the most carcinogenic effects of all the PAHs, has multiple toxic effects on marine bivalves. We investigated the interference mechanism of B[a]P on food metabolism (sugars, proteins, and sugars), and on reproductive endocrine and ovarian development in female scallops (Chlamys farreri). Scallops were exposed to different concentrations of B[a]P concentrations of 0, 0.38, 3.8, and 38 µg/L throughout gonadal development. Total cholesterol and triglyceride contents in the digestive glands were increased, and their synthesis genes were upregulated. The plasma glucose contents decreased with the inhibition of glycogen synthesis genes and the induction of glycolysis genes in the digestive gland. The results showed that B[a]P had endocrine-disrupting effects on scallops, that it negatively affected genes related to ovarian cell proliferation, sex differentiation, and egg development, and that it caused damage to ovarian tissue. Our findings supplement the information on B[a]P disruption in gonadal development of marine bivalves. Environ Toxicol Chem 2024;43:748-761. © 2023 SETAC.

Interactive effects of multiple antibiotic residues and ocean acidification on physiology and metabolome of the bay scallops Argopecten irradians irradians.

Coastal areas are confronted with compounding threats arising from both climatic and non-climatic stressors. Antibiotic pollution and ocean acidification are two prevalently concurrent environmental stressors. Yet their interactive effects on marine biota have not been investigated adequately and the compound hazard remain obscure. In this study, bay scallops Argopecten irradians irradians were exposed to multiple antibiotics (sulfamethoxazole, tetracycline, oxytetracycline, norfloxacin, and erythromycin, each at a concentration of 1 µg/L) combined with/without acidic seawater (pH 7.6) for 35 days. The single and interactive effects of the two stressors on A. irradians irradians were determined from multidimensional bio-responses, including energetic physiological traits as well as the molecular underpinning (metabolome and expressions of key genes). Results showed that multiple antibiotics predominantly enhanced the process of DNA repair and replication via disturbing the purine metabolism pathway. This alternation is perhaps to cope with the DNA damage induced by oxidative stress. Ocean acidification mainly disrupted energy metabolism and ammonia metabolism of the scallops, as evidenced by the increased ammonia excretion rate, the decreased O:N ratio, and perturbations in amino acid metabolism pathways. Moreover, the antagonistic effects of multiple antibiotics and ocean acidification caused alternations in the relative abundance of neurotransmitter and gene expression of neurotransmitter receptors, which may lead to neurological disorders in scallops. Overall, the revealed alternations in physiological traits, metabolites and gene expressions provide insightful information for the health status of bivalves in a natural environmental condition under the climate change scenarios.

Comparative study of domoic acid accumulation, isomer content and associated digestive subcellular processes in five marine invertebrate species.

Despite the deleterious effects of the phycotoxin domoic acid (DA) on human health, and the permanent threat of blooms of the toxic Pseudo-nitzschia sp. over commercially important fishery-resources, knowledge regarding the physiological mechanisms behind the profound differences in accumulation and depuration of this toxin in contaminated invertebrates remain very scarce. In this work, a comparative analysis of accumulation, isomer content, and subcellular localization of DA in different invertebrate species was performed. Samples of scallops Pecten maximus and Aequipecten opercularis, clams Donax trunculus, slippersnails Crepidula fornicata, and seasquirts Asterocarpa sp. were collected after blooms of the same concentration of toxic Pseudo-nitzschia australis. Differences (P < 0.05) in DA accumulation were found, wherein P. maximus showed up to 20-fold more DA in the digestive gland than the other species. Similar profiles of DA isomers were found between P. maximus and A. opercularis, whereas C. fornicata was the species with the highest biotransformation rate (∼10 %) and D. trunculus the lowest (∼4 %). DA localization by immunohistochemical analysis revealed differences (P < 0.05) between species: in P. maximus, DA was detected mainly within autophagosome-like vesicles in the cytoplasm of digestive cells, while in A. opercularis and C. fornicata significant DA immunoreactivity was found in post-autophagy residual bodies. A slight DA staining was found free within the cytoplasm of the digestive cells of D. trunculus and Asterocarpa sp. The Principal Component Analysis revealed similarities between pectinids, and a clear distinction of the rest of the species based on their capabilities to accumulate, biotransform, and distribute the toxin within their tissues. These findings contribute to improve the understanding of the inter-specific differences concerning the contamination-decontamination kinetics and the fate of DA in invertebrate species.

Biochemical response and tissue-specific accumulation of scallop Aequipecten tehuelchus from Patagonia, Argentina after exposure to inorganic arsenic.

This study investigates the effects of different inorganic arsenic (As III) concentrations (0, 125, 500 and 1000 µg As/L) following two exposure times (7 and 14 days) on gills, digestive gland and muscle of scallop Aequipecten tehuelchus from Patagonia, Argentina. A biochemical approach was used to investigate oxidative stress-related parameters after different As concentrations and exposure times. Although the accumulation of As was of the same order of magnitude in all tissues, the results showed distinct tissue-specific oxidative responses to this metalloid. Furthermore, the variation in exposure time had no significant effect on As accumulation in any of the three tissues. In gills, despite no reactive oxygen and nitrogen species (RONS) were detected, there was an increase in catalase (CAT) activity and metallothionein (MT) levels. Conversely, digestive gland showed RONS production without a rise in CAT and glutathione S-transferases (GST) activities, but with an increase in MT levels. In muscle, RONS production and CAT activity kept constant or decreased, while MT levels remained unchanged. In addition, exposure time demonstrated its critical role in gills by influencing the response of CAT, GST and MT, particularly at high As concentrations, while exposure time did not affect the biochemical stress parameters in the digestive gland and muscle. Interestingly, neither concentration of As produced lipid damage, showing the effectiveness of the antioxidant mechanisms to avoid it. These results emphasize that A. tehuelchus exhibited no time-dependent effects in response to As exposure, while showing tissue-specific responses characterized by significant concentration-dependent effects of As. This study provides a comprehensive insight by considering the combined effects of time and concentration of a contaminant and distinguishing its effects on specific tissues, a dimension often overlooked in the existing literature. Subsequent studies should prioritize the analysis of additional contaminants in species with increased sensitivity.

Whole-Genome Re-sequencing and Transcriptome Reveal Candidate Genes and Pathways Associated with Hybrid Sterility in Hermaphroditic Argopecten Scallops.

The interspecific hybrid scallops generated from the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) showed significant heterosis in growth. However, its sterility limits large-scale hybridization and hinders the development of the scallop breeding industry. Hybrid sterility is regulated by plenty of genes and involves a range of biochemical and physiological transformations. In this study, whole-genome re-sequencing and transcriptomic analysis were performed in sterile and fertile hybrid scallops. The potential genetic variations and abnormally expressed genes were detected to explore the mechanism underlying hybrid sterility in hermaphroditic Argopecten scallops. Compared with fertile hybrids, 24 differentially expressed genes (DEGs) with 246 variations were identified to be related to fertility regulation, which were mainly enriched in germarium-derived egg chamber formation, spermatogenesis, spermatid development, mismatch repair, mitotic and meiotic cell cycles, Wnt signaling pathway, MAPK signaling pathway, calcium modulating pathway, and notch signaling pathway. Specifically, variation and abnormal expression of these genes might inhibit the progress of mitosis and meiosis, promote cell apoptosis, and impede the genesis and maturation of gametes in sterile hybrid scallops. Eleven DEGs (XIAP, KAZN, CDC42, MEIS1, SETD1B, NOTCH2, TRPV5, M- EXO1, GGT1, SBDS, and TBCEL) were confirmed by qRT-PCR validation. Our findings may enrich the determination mechanism of hybrid sterility and provide new insights into the use of interspecific hybrids for extensive breeding.

The involvement of AMP-activated protein kinase α in regulating glycolysis in Yesso scallop Patinopecten yessoensis under high temperature stress.

AMP-activated protein kinase α subunit (AMPKα), the central regulatory molecule of energy metabolism, plays an important role in maintaining energy homeostasis and helping cells to resist the influence of various adverse factors. In the present study, an AMPKα was identified from Yesso scallop Patinopecten yessoensis (PyAMPKα). The open reading frame (ORF) of PyAMPKα was of 1599 bp encoding a putative polypeptide of 533 amino acid residues with a typical KD domain, a α-AID domain and a α-CTD domain. The deduced amino acid sequence of PyAMPKα shared 59.89-74.78% identities with AMPKαs from other species. The mRNA transcripts of PyAMPKα were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in adductor muscle. PyAMPKα was mainly located in cytoplasm of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyAMPKα, the phosphorylation level of PyAMPKα at Thr170 and the lactic acid (LD) content in adductor muscle all increased significantly, while the glycogen content decreased significantly. The activity of pyruvate kinase (PyPK) and the relative mRNA expression level of phosphofructokinase (PyPFK) were significantly up-regulated at 3 h after high temperature stress treatment (25 °C). Furthermore, the PyAMPKα activator AICAR could effectively upregulate the phosphorylation level of PyAMPKα, and increase activities of PyPFK and pyruvate kinase (PyPK). Meanwhile the glycogen content also declined under AICAR treatment. These results collectively suggested that PyAMPKα was involved in the high temperature stress response of scallops by enhancing glycolysis pathway of glycogen. These results would be helpful for understanding the functions of PyAMPKα in maintaining energy homeostasis under high temperature stress in scallops.

Mitochondria of Live Mizuhopecten yessoensis Scallops Can Sensitively Respond to Quality Changes during Dry/Reimmersed Storage as Determined by TMT-Labeled Proteomic Analysis.

Dry/reimmersed storage is often used in the transportation of live scallops. In this study, tandem mass tag (TMT)-labeled protein omics were used to quantitatively analyze the protein changes in scallops during dry/reimmersed stress. The results showed that during dry storage, scallops maintained cellular redox homeostasis through the upregulation of SCO1-like protein and thioredoxin domain-containing protein and reduced organic acids from the ATP synthetic process by the downregulation of NADH dehydrogenase, thereby reducing the damage caused during dry storage. During reimmersed storage, mitochondrial proteins underwent very sensitive changes. By upregulating aerobic respiration-related proteins (including proteins involved in glucose phosphate metabolism, glyceraldehyde 3-phosphate metabolism, etc.), the ATP synthesis ability was improved. However, the damage to the mitochondrial structure by dry storage could not be completely recovered, even by reimmersion. This included some apoptosis-related proteins that were obviously upregulated. In summary, compared with ATP-related indexes, mitochondria can respond more sensitively to dry storage stress.

Effect of high temperature stress on glycogen metabolism in gills of Yesso scallop Patinopecten yessoensis.

Glycogen is the main energy storage material in mollusc, and the regulation of its metabolism is essential for the response against high temperature stress. In the present study, the alternation of lactic acid (LD) content, glycogen reserves, mRNA expression level of genes encoding glycogen metabolism enzymes and activities of glycogen metabolism enzymes in gills of Yesso scallop Patinopecten yessoensis after an acute high temperature treatment at 25 °C were examined to understand the effect of high temperature on glycogen metabolism. The activity of T-ATPase in gills of scallops presented a gradual increase trend especially at 6 h after the acute high temperature treatment (p < 0.05). The glycogen reserves did not change significantly even there was a downward trend at 24 h after the acute high temperature treatment (p > 0.05). The mRNA transcripts of glycogen synthase (PyGCS) in gills of scallops decreased significantly at 1, 3, 6 and 12 h (p < 0.05), and recovered to normal level at 24 h (p > 0.05) after the acute high temperature treatment, while that of glycogen phosphorylase a (PyGPa) and phosphoenol pyruvate carboxy kinase (PyPEPCK) were both significantly down-regulated from 1 h to 24 h (p < 0.05) after the acute high temperature treatment. The activity of PyGPa at 1, 12 and 24 h and the content of LD at 3 and 24 h in gills of scallops after the acute high temperature treatment both increased significantly (p < 0.05). Furthermore, the mRNA transcripts of hexokinase (PyHK) and pyruvate kinase (PyPK) in gills of scallops increased significantly (p < 0.05) after the acute high temperature treatment, and the response of PyHK was stronger. However, there was no significant difference on the activity of PyPK in gills of scallops between the experimental samples and the blank samples (p > 0.05). In addition, the mRNA transcripts of citrate synthase (PyCS) in gills of scallops were significantly down-regulated at 6 h and 12 h (p < 0.05), and finally returned to normal level at 24 h (p > 0.05) after the acute high temperature treatment. These results collectively indicated acute high temperature stress leaded the alternation of glycogen metabolism in the gills of Yesso scallop, glycogenesis, gluconeogenesis and TCA cycle were inhibited, and the glycolysis pathway of glycogen was enhanced to produce more energy for coping with environmental pressure.

Cardiac performance and heart gene network provide dynamic responses of bay scallop Argopecten irradians irradians exposure to marine heatwaves.

The increased frequency of marine heat waves (MHWs) caused by global climate change is predicted to threaten the survival of economic bivalves, therefore having severely adverse effects on local ecological communities and aquaculture production. However, the study of scallops facing MHWs is still scarce, particularly in the scallop Argopecten irradians irradians, which has a significant share of "blue foods" in northern China. In the present study, bay scallop heart was selected to detect its cardiac performance, oxidative impairment and dynamic molecular responses, accompanied by assessing survival variations of individuals in the simulated scenario of MWHs (32 °C) with different time points (0 h, 6 h, 12 h, 24 h, 3 d, 6 d and 10 d). Notably, cardiac indices heart rate (HR), heart amplitude (HA), rate-amplitude product (RAP) and antioxidant enzyme activities superoxide dismutase (SOD) and catalase (CAT) all peaked at 24 h but sharply dropped on 3 d, coinciding with mortality. Transcriptome analysis revealed that the heart actively defended against heat stress at the acute stage (<24 h) via energy supply, misfolded proteins correction and enhanced signal transduction, whereas regulation of the defense response and apoptotic process combined with twice transcription initiation were the dominant responses at the chronic stage (3-10 d). In particular, HSP70 (heat shock protein 70), HSP90 and CALR (calreticulin) in the endoplasmic reticulum were identified as the hub genes (top 5 %) in the HR-associated module via WGCNA (weighted gene co-expression network analysis) trait-module analysis, followed by characterization of their family members and diverse expression patterns under heat exposure. Furthermore, RNAi-mediated knockdown of CALR expression (after 24 h) significantly weakened the thermotolerance of scallops, as evidenced by a drop of 1.31 °C in ABT (Arrhenius break temperature) between the siRNA-injected group and the control group. Our findings elucidated the dynamic molecular responses at the transcriptome level and verified the cardiac functions of CALR in bay scallops confronted with stimulated MHWs.

Integrated lipidomics and transcriptomics analysis reveal lipid metabolism disturbance in scallop (Chlamys farreri) exposure to benzo[a]pyrene.

Benzo[a]pyrene (B[a]P) commonly bioaccumulates in lipid-rich tissues due to its lipophilicity and further affects lipid metabolism. The present study systematically investigated the lipid metabolism disturbance in digestive glands of scallops (Chlamys farreri) exposure to B[a]P, based on lipidomics, transcriptomics, molecular and biochemical analysis. We exposed the scallops to environmentally relevant concentrations of B[a]P for 21 days. The bioaccumulation of B[a]P, lipid content and lipid peroxidation in digestive glands were measured. Integrated lipidomics and transcriptomics analysis, the differential lipid species were identified and key genes based on the pathways in which genes and lipid species involved together were selected in scallop exposure to 10 µg/L B[a]P. The changes of lipid profile showed that triglycerides (TGs) were accumulated after 21 days exposure, while the phospholipids (PLs) decreased demonstrated membrane structures were disrupted by B[a]P. In combination with the change of gene expression, we speculated that B[a]P could induce lipids accumulation by up-regulating lipid synthesis-related genes expression, down-regulating lipolysis-related genes expression and interfering with lipid transport. Overall, this study provides new insights into the mechanisms of lipid metabolism disturbance in bivalves exposed to PAHs, and establishes a foundation for understanding the bioaccumulation mechanism of B[a]P in aquatic organisms, which is of great importance for further ecotoxicological study.

Sources, bioaccumulation, and toxicity mechanisms of cadmium in Chlamys farreri.

The Potentially toxic elements (PTEs) cadmium (Cd) is one of the most serious stressors polluting the marine environment. Marine bivalves have specific high enrichment capacity for Cd. Previous studies have investigated the tissue distribution changes and toxic effects of Cd in bivalves, but the sources of Cd enrichment, migration regulation during growth, and toxicity mechanisms in bivalves have not been fully explained. Here, we used stable-isotope labeling to investigate the contributions of Cd from different sources to scallop tissues. We sampled the entire growth cycle of Chlamys farreri, which is widely cultured in northern China, from juveniles to adult scallops. We found tissue variability in the bioconcentration-metabolism pattern of Cd in different bound states, with Cd in the aqueous accounting for a significant contribution. The accumulation pattern of Cd in all tissues during growth was more significant in the viscera and gills. Additionally, we combined a multi-omics approach to reveal a network of oxidative stress-induced toxicity mechanisms of Cd in scallops, identifying differentially expressed genes and proteins involved in metal ion binding, oxidative stress, energy metabolism, and apoptosis. Our findings have important implications for both ecotoxicology and aquaculture. They also provide new insights into marine environmental assessment and mariculture development.