Dephosphorylation of T517 on Hemocyanin Is Required for Antibacterial Activity in Penaeus vannamei.

Posttranslational modifications expand the functions of immune-related proteins, especially during infections. The respiratory glycoprotein, hemocyanin, has been implicated in many other functions, but the role of phosphorylation modification in its functional diversity is not fully understood. In this study, we show that Penaeus vannamei hemocyanin (PvHMC) undergoes phosphorylation modification during bacterial infection. Dephosphorylation of PvHMC mediated by P. vannamei protein phosphatase 2A catalytic increases its in vitro antibacterial activity, whereas phosphorylation by P. vannamei casein kinase 2 catalytic subunit α decreases its oxygen-carrying capacity and attenuates its in vitro antibacterial activity. Mechanistically, we show that Thr517 is a critical phosphorylation modification site on PvHMC to modulate its functions, which when mutated attenuates the action of P. vannamei casein kinase 2 catalytic subunit α and P. vannamei protein phosphatase 2A catalytic, and hence abolishes the antibacterial activity of PvHMC. Our results reveal that phosphorylation of PvHMC modulates its antimicrobial functions in penaeid shrimp.

The evolutionary adaptation of shrimp hemocyanin subtypes and the consequences on their structure and functions.

Hemocyanin is the main respiratory protein of arthropods and is formed by hexameric and/or oligomeric subunits. Due to changes in the living environment and gene rearrangement, various hemocyanin subtypes and subunits evolved in crustaceans. This paper reviews the various hemocyanin subtypes and isoforms in shrimp and analyses published genomic data of sixteen hemocyanin family genes from Litopenaeus vannamei to explore the evolution of hemocyanin genes, subunits, and protein structure. Analysis of hemocyanin subtypes distribution and structure in various tissues was also performed and related to multiple and tissue-specific functions, i.e., immunological activity, immune signaling, phenoloxidase activity, modulation of microbiota homeostasis, and energy metabolism. The functional diversity of shrimp hemocyanin due to molecular polymorphism, transcriptional regulation, alternative splicing, degradation into functional peptides, interaction with other proteins or genes, and structural differences will also be highlighted for future research. Inferences would be drawn from other crustaceans to explain how evolution has changed the structure-function of hemocyanin and its implication for evolutionary research into the multifunctionality of hemocyanin and other related proteins in shrimp.

Deacetylation of K481 and K484 on Penaeid Shrimp Hemocyanin Is Critical for Antibacterial Activity.

Although invertebrates' innate immunity relies on several immune-like molecules, the diversity of these molecules and their immune response mechanisms are not well understood. Here, we show that Penaeus vannamei hemocyanin (PvHMC) undergoes specific deacetylation under Vibrio parahaemolyticus and LPS challenge. In vitro deacetylation of PvHMC increases its binding capacity with LPS and antibacterial activity against Gram-negative bacteria. Lysine residues K481 and K484 on the Ig-like domain of PvHMC are the main acetylation sites modulated by the acetyltransferase TIP60 and deacetylase HDAC3. Deacetylation of PvHMC on K481 and K484 allows PvHMC to form a positively charged binding pocket that interacts directly with LPS, whereas acetylation abrogates the positive charge to decrease PvHMC-LPS attraction. Besides, V. parahaemolyticus and LPS challenge increases the expression of Pvhdac3 to induce PvHMC deacetylation. This work indicates that, during bacterial infections, deacetylation of hemocyanin is crucial for binding with LPS to clear Gram-negative bacteria in crustaceans.

Synthetic microbial communities: Novel strategies to enhance the quality of traditional fermented foods.

Consumers are attracted to traditional fermented foods due to their unique flavor and nutritional value. However, the traditional fermentation technique can no longer accommodate the requirements of the food industry. Traditional fermented foods produce hazardous compounds, off-odor, and anti-nutritional factors, reducing product stability. The microbial system complexity of traditional fermented foods resulting from the open fermentation process has made it challenging to regulate these problems by modifying microbial behaviors. Synthetic microbial communities (SynComs) have been shown to simplify complex microbial communities and allow for the targeted design of microbial communities, which has been applied in processing traditional fermented foods. Herein, we describe the theoretical information of SynComs, particularly microbial physiological processes and their interactions. This paper discusses current approaches to creating SynComs, including designing, building, testing, and learning, with typical applications and fundamental techniques. Based on various traditional fermented food innovation demands, the potential and application of SynComs in enhancing the quality of traditional fermented foods are highlighted. SynComs showed superior performance in regulating the quality of traditional fermented foods using the interaction of core microorganisms to reduce the hazardous compounds of traditional fermented foods and improve flavor. Additionally, we presented the current status and future perspectives of SynComs for improving the quality of traditional fermented foods.

p53 Ubiquitination Comediated by HUWE1 and TRAF6 Contributes to White Spot Syndrome Virus Infection in Crustacean.

p53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection. IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean.

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.

Lacticaseibacillus paracasei fermentation broth identified peptide, Y2Fr, and its antibacterial activity on Vibrio parahaemolyticus.

Although Vibrio parahaemolyticus infections cause severe diseases of large yellow croaker (Larimichthys crocea), using antibiotics and other chemical agents to treat these infections could result in antimicrobial resistance, environmental pollution, and other associated problems. This study identified seven peptides from Lacticaseibacillus paracasei fermentation broth using ultra-high-performance liquid chromatography-mass spectrometry and screened antimicrobial peptide Y2Fr (VEIKNGLLKLNGKPLLIR) through its net charge, hydrophobicity and predicted secondary structure. Antibacterial activity analysis revealed that Y2Fr had a minimum inhibitory concentration (MIC) of 125 µg/mL, minimum bactericidal concentration (MBC) of 250 µg/mL against V. parahaemolyticus and a time-kill of 3 h. In a bacterial membrane environment, the secondary structure of peptide Y2Fr changed from a random coil to a ß-sheet to enhance its membrane permeability and binding to bacteria DNA to exert its antibacterial effect. Further molecular docking analysis revealed that peptide Y2Fr could bind to the membrane protein KKI11460.1 and DNA polymerase A0A0L8TVA4 of V. parahaemolyticus through hydrogen bonds. Meanwhile, treatment of Y2Fr with mammalian red blood cells and plasma revealed that it was noncytotoxic, nonhemolytic, and stable under physiological conditions. Thus, peptide Y2Fr has great potential use in treating and preventing infections caused by V. parahaemolyticus or similar bacteria in aquatic animals.

The Microbial Composition of Penaeid Shrimps' Hepatopancreas Is Modulated by Hemocyanin.

Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp.

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

LvHemB1, a novel cationic antimicrobial peptide derived from the hemocyanin of Litopenaeus vannamei, induces cancer cell death by targeting mitochondrial voltage-dependent anion channel 1.

Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.

Ammonia stress affects the structure and function of hemocyanin in Penaeus vannamei.

Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on aquaculture organisms such as penaeid shrimp because it makes them more susceptible to infections. In this study, we explored the effects of ammonia stress (0, 50, 100, and 150 mg/L) on the molecular structure and functions of the multifunctional respiratory protein hemocyanin (HMC) in Penaeus vannamei. While the mRNA expression of Penaeus vannamei hemocyanin (PvHMC) was up-regulated after ammonia stress, both plasma hemocyanin protein and oxyhemocyanin (OxyHMC) levels decreased. Moreover, ammonia stress changed the molecular structure of hemocyanin, modulated the expression of protein phosphatase 2 A (PP2A) and casein kinase 2α (CK2α) to regulate the phosphorylation modification of hemocyanin, and enhanced its degradation into fragments by trypsin. Under moderate ammonia stress conditions, hemocyanin also undergoes glycosylation to improve its in vitro antibacterial activity and binding with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Staphylococcus aureus) bacteria, albeit differently. The current findings indicate that P. vannamei hemocyanin undergoes adaptive molecular modifications under ammonia stress enabling the shrimp to survive and counteract the consequences of the stress.

Antimicrobial activity and acting mechanism of Tegillarca granosa hemoglobin-derived peptide (TGH1) against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a Gram-negative bacterium and the one of leading causal agent of human foodborne diseases such as gastroenteritis upon consumption of raw, or contaminated marine products. There is an increased interest in the use of antimicrobial peptides (AMPs) as alternative food preservatives to prevent foodborne diseases. In this study, bioinformatics tools were used to predict and screen AMPs derived from hemoglobin of blood clam (Tegillarca granosa). A novel AMP, T. granosa hemoglobin-derived peptide (TGH1), was identified and its antimicrobial effect and mechanism of action on V. parahaemolyticus was explored. The minimal inhibitory concentration (MIC) of TGH1 on V. parahaemolyticus was 12.5 µg/mL. Transmission electron microscopy (TEM) revealed that TGH1 kills bacteria by perforating the cell wall perforation and destroying integrity of the cell membrane. Similarly, laser confocal microscopy confirmed that TGH1 entered bacterial cells by aggregating on the cell surface to destroy the cell. In addition, TGH1 increased the inner-membrane permeability of V. parahaemolyticus in a concentration-dependent manner, as well as prevented biofilm formation. Moreover, TGH1 has 55.6% ß-sheet (antiparallel) structure and has no cytotoxic effects on normal human hepatocytes. Thus, peptide TGH1 has good potential use and application in antimicrobial control of foodborne pathogens.

The krüppel-like factor of Penaeus vannamei negatively regulates transcription of the small subunit hemocyanin gene as part of shrimp immune response.

Hemocyanin is a multifunctional respiratory glycoprotein, which has also been implicated in other biological functions in shrimp. Moreover, recent studies have revealed that hemocyanin is also involved in a broad range of immune-related activities in shrimp. However, in spite of the considerable interest in unraveling the reasons behind the multiple immune-related functions of hemocyanin, little is known about its transcriptional regulation. Here, DNA pull-down and Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) analyses were used to isolate and identify the putative transcription factor(s) that are involved in the transcriptional regulation of the small subunit hemocyanin gene of Penaeus vannamei (PvHMCs). Krüppel-like factor (designated PvKruppel), a zinc finger transcription factor homolog in P. vannamei, was identified among the putative transcription factors, while bioinformatics analysis revealed the presence of Krüppel-like factor binding site (KLF motif) on the core promoter region of PvHMCs. Mutational analysis and electrophoretic mobility shift assay (EMSA) confirmed that PvKruppel could bind to the KLF motif on the core promoter region of PvHMCs. Moreover, in response to lipopolysaccharide (LPS), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge, transcript levels of PvKruppel and PvHMCs were negatively correlated. Furthermore, overexpression of PvKruppel significantly reduced the promoter activity of PvHMCs, while PvKruppel knockdown by RNA interference or lipopolysaccharides (LPS) stimulation resulted in a significant increase in the transcript level of PvHMCs. Taken together, our present study provides mechanistic insights into the transcriptional regulation of PvHMCs by PvKruppel during shrimp immune response to pathogens.

Litopenaeus vannamei sirtuin 6 homolog (LvSIRT6) is involved in immune response by modulating hemocytes ROS production and apoptosis.

The histone deacetylase, sirtuin 6 (SIRT6), plays an essential role in the regulation of oxidative stress, mitochondrial function and inflammation in mammals. However, the specific role of SIRT6 in invertebrate immunity has not been reported. Here, we characterized for the first time, a sirtuin 6 homolog in Litopenaeus vannamei (LvSIRT6), with full-length cDNA of 2919 bp and 1536 bp open reading frame (ORF) encoding a putative protein of 511 amino acids, which contains a typical SIR2 domain. Sequence and phylogenetic analysis revealed that LvSIRT6 shares a close evolutionary relationship with SIRT6 from invertebrates. Real-time quantitative PCR analysis of LvSIRT6 transcripts revealed that they were ubiquitously expressed in shrimp and induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting the involvement of LvSIRT6 in shrimp immune response. Moreover, knockdown of LvSIRT6 decreased mitochondrial membrane potential and increased total ROS level in hemocytes, especially upon V. parahaemolyticus challenge. Depletion of LvSIRT6 also increased hemocytes apoptosis in terms of decreased expression of pro-survival LvBcl-2, but increased expression of pro-apoptotic LvBax and LvCytochrome C, coupled with high LvCaspase3/7 activity. Shrimp were rendered more susceptible to V. parahaemolyticus infection upon LvSIRT6 knockdown. Taken together, our present data suggest that LvSIRT6 plays an important role in shrimp immune response by modulating hemocytes ROS production and apoptosis during pathogen challenge.

The TOR pathway participates in the regulation of growth development in juvenile spotted drum (Nibea diacanthus) under different dietary hydroxyproline supplementation.

Commonly used aquatic feed naturally contains low-level or no hydroxyproline (Hyp). This study was conducted to evaluate the effects of dietary Hyp inclusion on growth performance, body composition, amino acid profiles, blood biochemistry, and the expression of target of rapamycin (TOR) pathway-related genes in juvenile Nibea diacanthus. Fish with similar size (initial body weight, 133.00 ± 2.14 g) were fed six isonitrogenous and isolipidic practical diets supplemented with graded levels of Hyp (0, 5, 10, 15, 20, and 25 g kg-1 of dry matter) for 8 weeks. The results indicated that growth performance and feed utilization were improved with increased levels of dietary Hyp (P < 0.05), and the optimum amount of dietary Hyp estimated from SGR as 16.6 g kg-1. The crude protein of whole body and swim bladder and the amino acid composition of muscle and swim bladder were significantly (P < 0.05) affected by the addition of dietary Hyp, which reflects the important role of feed composition in animal body composition. In addition, the expression levels of mammalian target of rapamycin (TOR) and ribosomal protein S6 kinase1 (S6K1) genes in the liver, muscle, and swim bladder increased with increasing Hyp content of diets, while the mRNA expression level of eukaryotic translation initiation factor 4E-binding protein (4E-BP) gene in these tissues decreased. These results indicated that Hyp improved fish growth and the ability to synthesize proteins, most likely through the TOR pathway. It is suggested that dietary Hyp supplementation is particularly necessary for application in aquatic feed.

Litopenaeus vannamei Src64B restricts white spot syndrome virus replication by modulating apoptosis.

The Src family kinases (SFK) are involved in signaling transductions that regulate numerous biological activities including host-virus interaction. These features of SFK have been well explored in vertebrates, however, in shrimp, the invertebrate SFK family member Src64B, has not been characterized and therefore its role in shrimp-virus interaction remains unknown. In this study, two Litopenaeus vannamei Src64B isoforms (designated LvSrc64B1 and LvSrc64B2) were first cloned and their role in white spot syndrome virus (WSSV) infection was explored. Bioinformatics analysis revealed that LvSrc64B1 and LvSrc64B2 were similar to other Src64B family members, with high homology in primary and tertiary structures, and contained the conserved SFK functional domains, as well as the putative myristylation and phosphorylation sites. Tissue distribution analysis showed that both LvSrc64B isoforms were ubiquitously expressed, albeit distinctively in the tested tissues. In addition, transcript levels of LvSrc64B1 and LvSrc64B2 were significantly induced following WSSV challenge and had similar expression patterns. Furthermore, siRNA-mediated knockdown of LvSrc64B1 and LvSrc64B2 followed by WSSV infection resulted in increased expression of viral genes, enhanced viral DNA replication, and elevation of hemocytes apoptosis. Depletion of LvSrc64B1 and LvSrc64B2 also reduced shrimp survival upon WSSV infection. In conclusion, the current data strongly suggest that Src64B is a host factor that inhibits WSSV replication by modulating apoptosis in shrimp.

Litopenaeus vannamei CK2 is involved in shrimp innate immunity by modulating hemocytes apoptosis.

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2ß) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2ß has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2ß transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.

c-Jun regulates the promoter of small subunit hemocyanin gene of Litopenaeus vannamei.

Hemocyanin (HMC) is a respiratory glycoprotein, which also plays multifunctional non-specific innate immune defense functions in shrimp. However, the transcriptional regulatory mechanisms of the hemocyanin gene expression have not been reported. In the present study, we cloned a 4324 bp fragment of small subunit hemocyanin (HMCs) gene of Litopenaeus vannamei including the 5'-flanking region, from upstream 2475 bp to downstream 1849 bp (exon 1-intron 1-exon 2) by genome walking method. Four deletion constructs were then generated and their promoter activity assessed using the luciferase reporter system. Interestingly, we identified an alternative promoter (+1516/+1849 bp) located in exon 2, which has stronger promoter activity than the full-length or the other constructs. Bioinformatics analyses revealed that the alternative promoter region contains two conserved binding sites of the transcription factor c-Jun. Mutational analysis and electrophoretic mobility shift assay showed that Litopenaeus vannamei c-Jun (Lvc-Jun) binds to the region +1582/+1589 bp and +1831/+1837 bp of the alternative promoter. Furthermore, overexpression of Lvc-Jun significantly increased the alternative promoter activity, while co-transfection with dsRNA-Lvc-Jun significantly reduced the alternative promoter activity of HMCs. Taken together, our present data indicate that the transcription factor Lvc-Jun is essential for the transcriptional regulation of the HMCs gene expression.

SpBAG1 promotes the WSSV infection by inhibiting apoptosis in mud crab (Scylla paramamosain).

Bcl-2 associated athanogene-1 (BAG1) is involved in various signalling pathways including apoptosis, cell proliferation, gene transcriptional regulation and signal transduction in animals. However the functions of BAG1 during the antiviral response of mud crab Scylla paramamosain is still unclear. In this study, the mud crab BAG1 (SpBAG1) was characterized to consist of 1761 nucleotides, containing an opening frame of 630bp encoding 209 amino acids with an ubiquitin domain and a BAG1 domain. SpBAG1 was found to be significantly up-regulated at 6 h-24 h, but down-regulated from 48 h-72 h in the hemocytes of mud crab after challenge with white spot syndrome virus (WSSV). RNAi knock-down of SpBAG1 significantly reduced the copies of WSSV and increased the apoptotic rate in mud crabs. The finding from this study suggested that SpBAG1 could promote the WSSV infection by inhibiting apoptosis in mud crab. Therefore, to the best of our knowledge, this is the first study demonstrating the role of SpBAG1 as a novel apoptosis inhibitor to promote virus infection in mud crab.

In-depth proteomic profiling of the Singapore grouper iridovirus virion.

Singapore grouper iridovirus (SGIV) is a lethal grouper virus containing 162 predicted ORFs. Previous proteomic studies led to identification of 73 SGIV structural proteins. Here, SDS-assisted tube-gel digestion and DOC-assisted in-solution digestion coupled with LC-ESI-MS/MS were applied to further profile the SGIV structural proteome. We identified a total of 90 SGIV structural proteins including 24 newly reported proteins. Additionally, several PTMs were identified, including 26 N-terminal acetylated proteins, three phosphorylated proteins, and one myristoylated protein. Importantly, 47 of the proteins that were identified are predicted to contain conserved domains. Our work greatly expands the repertoire of the SGIV structural proteome and provides more insight into the biology of SGIV.