Effects of high stocking density on growth performance and expression of MyD88, and its temporal expression under the challenge of Vibrio parahaemolyticus in the noble scallop Chlamys nobilis.

High stocking density has been regarded as an adverse factor in bivalve aquaculture. However, its subsequent molecular response to pathogenic bacteria has been little studied. In order to study the question, a novel MyD88 was first cloned using adult noble scallops Chlamys nobilis (CnMyD88), and its tissue distribution was investigated. Then, 1860 juvenile scallops were divided into two groups with two initial densities of high density (200 individuals/layer, HD) and normal density (110 individuals/layer, ND) and in-situ cultured for three months, in which their growth, survival, and the differential expression of CnMyD88 were examined, respectively. Finally, scallops were injected with the Vibrio parahaemolyticus to assess the temporal expression of CnMyD88. As the results show, CnMyD88 cDNA has a full length of 2241 bp and contains an 1107 bp ORF that encodes a 368-derived protein. It was widely expressed in examined tissues with a significantly higher level in hemolymph, intestine, mantle, and gonad than others. Besides, the HD group showed lower growth (0.39 ± 0.05 mm/day) and survival (37.00 ± 8.49%) than the ND group (0.55 ± 0.02 mm/day and 76.82 ± 5.78%). More importantly, the HD group exhibited significantly lower expression levels of CnMyD88 in their examined tissues than the ND group. After V. parahaemolyticus challenging, CnMyD88 had significantly lower expression levels in the scallops from the HD group than that of the scallops from the ND group at 6th, 24th, and 36th. The present results indicated that high stocking density not only made adverse impacts on growth and survival but also may induce immunosuppression in the noble scallop. Therefore, appropriate low stocking density may be worth considering to adopt in scallop aquaculture.

Endogenous LC-PUFA biosynthesis capability in commercially important mollusks.

Mollusks are excellent dietary sources for LC-PUFA. However, the main challenge limiting mollusk production is the high mortality rate of molluskan larvae in early life cycle stages. This paper reviews scientific evidences on molecular and biochemical studies of LC-PUFA biosynthesis in commercially important molluskan species. It carefully summarizes the pertinent data published on specific research questions to improve the understanding of the diverse evidences. It is helpful to clarify the current state of research and determine topics for future studies on LC-PUFA biosynthesis in mollusks. From the analysis of published data, mollusks have the ability to biosynthesis LC-PUFA to a certain extent. LC-PUFA biosynthesis information of commercially important molluskan species can be useful to determine the fatty acids essential for their diet. Therefore, specific management strategies or feeds can be developed to strengthen the industry by improving the health and survival rate of molluskan larvae.

Carotenoid content and composition: A special focus on commercially important fish and shellfish.

Carotenoids are natural pigments that provide many health benefits to living organisms. Although terrestrial plants are the major dietary source of carotenoids for humans, aquatic animals (especially fish and shellfish) are equally important because they are rich in certain important carotenoids lacking in fruits and vegetables. Although extensive research has focused on exploring the carotenoid content and composition in fish and shellfish, this information is poorly organized. This paper reviews the scientific evidence for the carotenoid content and composition in fish and shellfish. It makes serious attempts to summarize the relevant data published on specific research questions in order to improve the understanding of various evidence to clarify the research status of carotenoids in fish and shellfish and defining topics for future studies. From the analysis of published data, it is obvious that most fish and shellfish are rich in complex carotenoids (e.g. astaxanthin, fucoxanthin, fucoxanthinol, lutein). These carotenoids have stronger antioxidant effect, higher efficiency in removing the singlet oxygen and the peroxyl radicals, and have a variety of health benefits. Carotenoid levels in fish and shellfish depend on genotype, climatic conditions of the production area, storage and cooking methods. However, the information of the bioavailability of fish/shellfish carotenoids to human is very limited, which hinders the actual contributions to health. The findings of this study can be used as a guide to select appropriate fish and shellfish as dietary sources of carotenoids, and provide information about potential fish and shellfish species for aquaculture to produce carotenoids to meet part of the growing demand for natural carotenoids.

Lipid nutritional quality of marine and freshwater bivalves and their aquaculture potential.

Omega-3 Long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are beneficial to human health. Since the industrial revolution, with the tremendous increase of human population, the supply of natural n-3 LC-PUFA is far lower than the nutritional need of n-3 LC-PUFA. Therefore, a new alternative source of natural n-3 LC-PUFA is urgently needed to reduce the supply and demand gap of n-3 LC-PUFA. Mollusks, mainly bivalves, are rich in n-3 LC-PUFA, but the information of bivalves' lipid profile is not well organized. Therefore, this study aims to analyze the published fatty acid profiles of bivalves and reveal the potential of bivalve aquaculture in meeting the nutritional needs of human for n-3 LC-PUFA. There are growing evidence show that the nutritional quality of bivalve lipid is not only species-specific, but also geographical specific. To date, bivalve aquaculture has not been evenly practiced across the globe. It can be seen that aquaculture is predominant in Asia, especially China. Unlike fish aquaculture, bivalve aquaculture does not rely on fishmeal and fish oil inputs, so it has better room for expansion. In order to unleash the full potential of bivalve aquaculture, there are some challenges need to be addressed, including recurrent mass mortalities of farmed bivalves, food safety and food security issues. The information of this article is very useful to provide an overview of lipid nutritional quality of bivalves, and reveal the potential of bivalve aquaculture in meeting the growing demand of human for n-3 LC-PUFA.

Comparative profiling of ovarian and testicular piRNAs in the mud crab Scylla paramamosain.

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development.

Identification of male-specific SNP markers and development of PCR-based genetic sex identification technique in crucifix crab (Charybdis feriatus) with implication of an XX/XY sex determination system.

In this study, we first identified male-specific SNP markers using restriction site-associated DNA sequencing, and further developed a PCR-based sex identification technique for Charybdis feriatus. A total of 296.96 million clean reads were obtained, with 114.95 and 182.01 million from females and males. After assembly and alignment, 10 SNP markers were identified being heterozygous in males but homozygous in females. Five markers were further confirmed to be male-specific in a large number of individuals. Moreover, two male-specific sense primers and a common antisense primer were designed, using which, a PCR-based genetic sex identification method was successfully developed and used to identify the sex of 103 individuals, with a result of 49 females and 54 males. The presence of male-specific SNP markers suggests an XX/XY sex determination system for C. feriatus. These findings should be helpful for better understanding sex determination mechanism, and drafting artificial breeding program in crustaceans.

mRNA profile provides novel insights into stress adaptation in mud crab megalopa, Scylla paramamosain after salinity stress.

BACKGROUND: Mud crab, Scylla paramamosain, a euryhaline crustacean species, mainly inhabits the Indo-Western Pacific region. Wild mud crab spawn in high-salt condition and the salinity reduced with the growth of the hatching larvae. When the larvae grow up to megalopa, they migrate back to estuaries and coasts in virtue of the flood tide, settle and recruit adult habitats and metamorphose into the crablet stage. Adult crab can even survive in a wide salinity of 0-35 ppt. To investigate the mRNA profile after salinity stress, S. paramamosain megalopa were exposed to different salinity seawater (low, 14 ppt; control, 25 ppt; high, 39 ppt). RESULTS: Firstly, from the expression profiles of Na+/K+/2Cl- cotransporter, chloride channel protein 2, and ABC transporter, it turned out that the 24 h might be the most influenced duration in the short-term stress. We collected megalopa under different salinity for 24 h and then submitted to mRNA profiling. Totally, 57.87 Gb Clean Data were obtained. The comparative genomic analysis detected 342 differentially expressed genes (DEGs). The most significantly DEGs include gamma-butyrobetaine dioxygenase-like, facilitated trehalose transporter Tret1, sodium/potassium-transporting ATPase subunit alpha, rhodanese 1-like protein, etc. And the significantly enriched pathways were lysine degradation, choline metabolism in cancer, phospholipase D signaling pathway, Fc gamma R-mediated phagocytosis, and sphingolipid signaling pathway. The results indicate that in the short-term salinity stress, the megalopa might regulate some mechanism such as metabolism, immunity responses, osmoregulation to adapt to the alteration of the environment. CONCLUSIONS: This study represents the first genome-wide transcriptome analysis of S. paramamosain megalopa for studying its stress adaption mechanisms under different salinity. The results reveal numbers of genes modified by salinity stress and some important pathways, which will provide valuable resources for discovering the molecular basis of salinity stress adaptation of S. paramamosain larvae and further boost the understanding of the potential molecular mechanisms of salinity stress adaptation for crustacean species.

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.

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.

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.

The miRNAs profiling revealed by high-throughput sequencing upon WSSV infection in mud crab Scylla paramamosain.

microRNAs (miRNAs) are known to regulate various immune functions by silencing the target genes in both vertebrates and invertebrates. However, in mud crab Scylla paramamosain, the role of miRNAs during the response to virus invasion remains unclear. To investigate the roles of miRNAs in S. paramamosain during virus infection, the mud crab was challenged with white spot syndrome virus (WSSV) and then subjected to the transcriptional analysis at different conditions. The results of high-throughput sequencing revealed that 940,379 and 1,306,023 high-quality mappable reads were detected in the hemocyte of normal and WSSV-infected mud crabs, respectively. Besides, the total number of 261 unique miRNAs were identified. Among them, 131 miRNAs were specifically expressed in the hemocytes of normal mud crabs, 46 miRNAs were specifically transcribed in those of WSSV-infected individuals, the other 84 miRNAs were expressed in both normal and WSSV-infected individuals. Furthermore, a number of 152 (89 down-regulated and 63 up-regulated) miRNAs were found to be differentially expressed in the WSSV-infected hemocytes, normalized to the controls. The identified miRNAs were subjected to GO analysis and target gene prediction and the results suggested that the differentially regulated miRNAs were mainly correlated with the changes of the immune responses of the hemocytes, including phagocytosis, melanism, and apoptosis as well. Taken together, the results demonstrated that the expressed miRNAs during the virus infection were mainly involved in the regulation of immunological pathways in mud crabs. Our findings not only enrich the understanding of the functions of miRNAs in the innate immune system but also provide some novel potential targets for the prevention of WSSV infection in crustaceans.

Climate Change and Bivalve Mass Mortality in Temperate Regions.

One of the fastest-growing global food sectors is the bivalve aquaculture industry. Bivalves particularly oysters, mussels and clams are important sources of animal protein (Tan and Ransangan 2016a, b). Bivalve aquaculture represents 14-16% of the average per capita animal protein for 1.5 billion people and supports over 200,000 livelihoods, mostly in developing countries (FAO 2018). Most of the bivalves produced around the world (89%) are from aquaculture (FAO 2016). To date, mollusc aquaculture have accounted for 21.42% (17.14 million tonnes) of the total aquaculture production, with Asia being the largest contributor (92.27%) (FAO 2018).

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.

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.

Two novel serine proteases from Scylla paramamosain involved in the synthesis of anti-lipopolysaccharide factors and activation of prophenoloxidase system.

Serine proteases (SPs) are important in various immune responses, including prophenoloxidase (proPO) activation, antimicrobial peptides (AMPs) synthesis, and hemolymph coagulation in invertebrates. In this study, SP3 and SP5 of mud crab (Scylla paramamosain) were studied. SP3 and SP5 were expressed in all examined tissues (mainly in hemocytes), and are associated with the immune responses of mud crab to Vibrio parahemolyticus and Staphylococcus aureus, as well as interacted with TRAF6, and are involved in the activation of anti-lipopolysaccharide factors (ALFs) probably through the TLR/NF-κB pathway. Depletion of SP3 inhibited the expression of ALF1, ALF2, ALF3, and ALF6, while knockdown of SP5 significantly decreased ALF5, and ALF6. Furthermore, both SP5 and TRAF6 regulated the PO activity in the hemolymph of mud crab. Overexpression assay showed that both SP3 and SP5 could enhance the promoter activities of ALFs in mud crab. Taken together, the results of this study indicate that SP3 and SP5 might play important roles in the immune system of mud crab against pathogen invasion.

Identification of two ferritin genes and their expression profiles in response to bacterial challenge in noble scallop Chlamys nobilis with different carotenoids content.

As a major intracellular iron storage protein, ferritin plays important roles in iron homeostasis and innate immunity. In this study, two novel ferritin subunits from noble scallop Chlamys nobilis (CnFer1 and CnFer2) were identified and analyzed. The open reading frame of CnFer1 and CnFer2 was 522 and 519bp long, encoding 173 and 172 amino acids, respectively. Both ferritins contained a putative iron-binding region signature (IBRS). Analysis of putative conserved domains showed the two CnFer genes contained three key domains of ferritin subunits, a ferroxidase diiron center (E25, Y32, E59, E60, H63, E105, and Q139), an iron ion channel (H116, D129, E132) and a ferrihydrite nucleation center (D58, E59, and E62) that present in M type subunits. A putative iron response element (IRE) was observed at both CnFer genes in the 5' UTR. Phylogenetic analysis result suggested that the two genes are cytoplasmic ferritins and have the closest evolution relationship with ferritins from Mizuhopecten yessoensis. The two ferritin genes were wildly expressed in examined tissues and the highest level was found in gill. After V. parahaemolyticus challenged, both CnFer genes were significantly up-regulated suggesting that they are important proteins involved in host immune defense. Moreover, under bacterial challenge, the expression levels of both two genes in Golden scallops (rich in carotenoids) were significantly higher than that in Brown scallops (less in carotenoids) which suggesting that carotenoids enhance the immunity in scallops to defense against the bacterial stress.

Innate immune responses and metabolic alterations of mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is one of the major pathogens caused diseases in cultured mud crab (Scylla paramamosain). Mud crabs lack an adaptive immune system, their defenses depend almost on innate immunity. Evaluation of the molecular responses of mud crabs to pathogens is essential for control of disease occurrence in farmed animals. In this study, the impacts of V. parahaemolyticus on immunity-related genes and metabolites in mud crabs of different groups (PG, SG and MG refer to controlled, survival and moribund groups, respectively) were investigated. Our results revealed that V. parahaemolyticus infection stimulated significant expressions of immune-related genes (prophenoloxidase, alpha 2-macroglobulin, lysosomal-associated membrane protein, Rab5, C-type lectin B and anti-lipopolysaccharide factor 5) in the MG within 72 h post-infection. The ATP content was significantly reduced in all tissues except muscle of moribund mud crabs. A total of 668 metabolites (including 190 down-regulated and 145 up-regulated) were identified and assigned to 77 pathways in both SG and MG. Metabolites involved in the saturated fatty acid are up-regulated, whereas unsaturated fatty acid and amino acid metabolisms are down-regulated in the immune system of mud crabs during the bacterial infection in MG. Furthermore, a reduction of hemocyte number and an increase of microbial abundance was found in MG. Our results demonstrated that V. parahaemolyticus induced death of mud crabs through reducing the metabolites associate with energy biosynthesis and innate immune system (i.e. proliferation of hemocyte and melanization), resulting in decrease of ATP in different tissues and failed to clearance of pathogens, respectively. The findings of this study provide a basic information of the responses of mud crab on bacterial infection, which is essential for prevention and control of diseases in mud crab aquaculture.

Effects of stocking density on the growth performance, bacterial load and antioxidant response systems of noble scallop Chlamys nobilis.

Stocking density is a crucial factor in shellfish aquaculture that affects overall growth performance and health status. Present study analyzes the effects of stocking densities on growth, survival and hemolymph immune status of noble scallop Chlamys nobilis. The scallops with the same size were separately placed in the lantern cages (10 layers per cage) using high stocking density (500 scallops per cage) and low stocking density (100 scallops per cage) and cultivated in the same location for 60 days. The results indicated that the scallops cultivated at high stocking density had significantly higher mortality and slower growth than those cultivated at low stocking density. Moreover, the hemolymph of scallops cultivated at high density showed significantly higher bacterial load, higher reactive oxygen species (ROS), higher expression level of Nrf2 and lower expression level of Keap1, as well as lower antibacterial ability of Vibrio parahemolyticus than that of scallops cultivated at low density. The present results demonstrated that long-term overcrowding is detrimental for the scallops, which can not only lead to high mortality and slow growth, but also cause more vulnerable to pathogenic bacteria. Therefore, we speculated that high stocking density culture practice of scallops in China might be the root of infectious bacteria outbreaks.

SpTGase plays an important role in the hemolymph clotting in mud crab (Scylla paramamosain).

Transglutaminase (TGase) is important in blood coagulation, a conserved immunological defense mechanism among invertebrates. This study is the first report of the TGase in mud crab (Scylla paramamosain) (SpTGase) with a 2304 bp ORF encoding 767 amino acids (molecular weight 85.88 kDa). SpTGase is acidic, hydrophilic, stable and thermostable, containing three transglutaminase domains, one TGase/protease-like homolog domain (TGc), one integrin-binding motif (Arg270, Gly271, Asp272) and three catalytic sites (Cys333, His401, Asp424) within the TGc. Neither a signal peptide nor a transmembrane domain was found, and the random coil is dominant in the secondary structure of SpTGase. Phylogenetic analysis revealed a close relation between SpTGase to its homolog EsTGase 1 from Chinese mitten crab (Eriocheir sinensis). Expression of SpTGase was investigated using qRT-PCR (1) in eight tissues from healthy mud crabs, with the highest expression in hemocytes, and (2) in response to various immune challenges (Vibrio parahaemolyticus, lipopolysaccharide (LPS) or Poly I:C infection), revealing a major up-regulation in hemocytes, skin, and hepatopancreas during the 96-h post injection. The recombinant SpTGase showed a capacity of agglutination activities on both Gram-negative bacteria and yeast. SpTGase was found to directly interact with another important blood coagulation component clip domain serine protease (SpcSP). Moreover, knockdown of SpTGase resulted in a decreased expression of both clotting protein precursor (SppreCP) and SpcSP and an increase of duration time in the blood coagulation. Taken together, the findings of this study suggest SpTGase play an important role in the hemolymph clotting in mud crab S. paramamosain.