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.

The transcription factor CEBP homolog of Penaeus vannamei contributes to WSSV replication.

The cellular transcription factors are known to play important roles in virus infection. The present study cloned and characterized a transcription factor CCAAT/Enhancer-binding protein homolog from the shrimp Penaeus vannamei (designates as PvCEBP), and explored its potential functions in white spot syndrome virus (WSSV) infection. PvCEBP has an open reading frame (ORF) of 864 bp encoding a putative protein of 287 amino acids, which contained a highly C-terminal conserved bZIP domain. Phylogenetic tree analysis showed that PvCEBP was evolutionarily clustered with invertebrate CEBPs and closely related to the CEBP of Homarus americanus. Quantitative real-time PCR (qPCR) analysis revealed that PvCEBP was expressed in all examined shrimp tissues, with transcript levels increased in shrimp hemocytes and gill upon WSSV challenge. Furthermore, knockdown of PvCEBP mediated by RNA interference significantly decreased the expression of WSSV genes and viral loads, while enhanced the shrimp survival rate under WSSV challenge. In silico prediction and reporter gene assays demonstrated that PvCEBP could activate the promoter activity of the viral immediate-early gene ie1. Collectively, our findings suggest that PvCEBP is annexed by WSSV to promote its propagation by enhancing the expression of viral immediate-early genes.

Modulating the Coordination of Single Co Atoms to Trigger the Catalytic Oxidation of Formaldehyde at Room Temperature.

Designing efficient and stable non-precious metal catalysts remains a significant challenge for formaldehyde (HCHO) oxidation, which is an expected way to replace the employment of noble-metal catalysts. Herein, a series of atomically dispersed Co catalysts are optimized by evaporating nitrogen atoms and exploring their HCHO oxidation catalytic performance. The results show that the prepared temperature can effectively control the coordination regulation of the Co atomic site, which in turn affects the catalytic oxidation activity. Our best catalyst, the Co-N/C prepared at 1000 °C, exhibits superior activity with 92.8% of conversion at room temperature at a gas hourly space velocity (GHSV) of 72,000 mL·g-1·h-1. Extensive characterizations combined with theoretical calculations reveal that the high catalytic activity is attributed to the low-coordinated center, which can be tailored by pyrolysis temperature. This work provides an innovative strategy for catalyst design in the catalytic oxidation reaction.

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.

Comparative Transcriptome Analysis Reveals That WSSV IE1 Protein Plays a Crucial Role in DNA Replication Control.

For DNA viruses, the immediate-early (IE) proteins are generally essential regulators that manipulate the host machinery to support viral replication. Recently, IE1, an IE protein encoded by white spot syndrome virus (WSSV), has been demonstrated to function as a transcription factor. However, the target genes of IE1 during viral infection remain poorly understood. Here, we explored the host target genes of IE1 using RNAi coupled with transcriptome sequencing analysis. A total of 429 differentially expressed genes (DEGs) were identified from penaeid shrimp, of which 284 genes were upregulated and 145 genes were downregulated after IE1 knockdown. GO and KEGG pathway enrichment analysis revealed the identified DEGs are significantly enriched in the minichromosome maintenance (MCM) complex and DNA replication, indicating that IE1 plays a critical role in DNA replication control. In addition, it was found that Penaeus vannamei MCM complex genes were remarkably upregulated after WSSV infection, while RNAi-mediated knockdown of PvMCM2 reduced the expression of viral genes and viral loads at the early infection stage. Finally, we demonstrated that overexpression of IE1 promoted the expression of MCM complex genes as well as cellular DNA synthesis in insect High-Five cells. Collectively, our current data suggest that the WSSV IE1 protein is a viral effector that modulates the host DNA replication machinery for viral replication.

Shrimp Lipid Droplet Protein Perilipin Involves in the Pathogenesis of AHPND-Causing Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by a unique strain of Vibrio parahaemolyticus (Vp (AHPND)), has become the world's most severe debilitating disease in cultured shrimp. Thus far, the pathogenesis of AHPND remains largely unknow. Herein, in Litopenaeus vannamei, we found that a Vp (AHPND) infection significantly increased the expression of lipid droplets (LDs) protein LvPerilipin, as well as promoted the formation of LDs. In addition, the knockdown of LvPerilipin increased the shrimp survival rate in response to the Vp (AHPND) infection, and inhibited the proliferation of Vp (AHPND). Furthermore, we demonstrated that LvPerilipin depletion could increase the production of reactive oxygen species (ROS), which may be responsible for the decreased Vp (AHPND) proliferation. Taken together, our current data for the first time reveal that the shrimp lipid droplets protein Perilipin is involved in the pathogenesis of Vp (AHPND) via promoting LDs accumulation and decreasing ROS production.

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.

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.

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.

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.

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.

Acute Hepatopancreatic Necrosis Disease (AHPND) related microRNAs in Litopenaeus vannamei infected with AHPND-causing strain of Vibrio parahemolyticus.

BACKGROUND: Acute hepatopancreatic necrosis disease (AHPND) has emerged as a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. Given that several diseases and infections have been associated with microRNAs (miRNAs), we conducted a comparative transcriptomic analysis using the AHPND (VA) and non-AHPND (VN) strains of Vibrio parahemolyticus to identify miRNAs potentially involved in AHPND pathogenesis in Litopenaeus vannamei. RESULTS: A total of 83 miRNAs (47 upregulated and 36 downregulated) were significantly differentially expressed between the VA and VN challenged groups, while 222 target genes of these miRNAs were predicted. Functional enrichment analysis revealed that the miRNAs target genes were involved in multiple biological processes including metabolic pathways, amoebiasis, Vibrio cholerae infection etc. Finally, interaction network and qPCR (Real-time Quantitative PCR) analysis of 12 potential key AHPND-related miRNAs and their predicted target genes, revealed their possible involvement in modulating several immune-related processes in the pathogenesis of AHPND. CONCLUSIONS: We have shown using comparative transcriptomic analysis, miRNAs and their target genes that are responsive to AHPND V. parahemolyticus infection in shrimp, therefore suggesting their possible role in defense response to AHPND V. parahemolyticus infection.

Identification and molecular characterization of the Pim1 serine/threonine kinase homolog in Litopenaeus vannamei.

The Pim1 serine/threonine kinase is associated with multiple cellular functions including proliferation, survival, differentiation, apoptosis, tumorigenesis, immune regulation and inflammation in vertebrates. However, little is known about the role of Pim1 in invertebrate immunity. In this study, we identified and characterized for the first time, a Pim1 (LvPim1) gene in Litopenaeus vannamei, with a full-length cDNA of 2352 bp and a 1119 bp open reading frame (ORF) encoding a putative protein of 372 amino acids, which contains a typical serine/threonine kinase domain. Sequence and phylogenetic analysis revealed that LvPim1 shared a close evolutionary relationship with Pim1 from vertebrates. Real-time qPCR analysis showed that LvPim1 was widely expressed in all tissues tested; with its transcript level induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), thus, suggesting its probable involvement in shrimp immune response. Moreover, knockdown of LvPim1 resulted in increased hemocytes apoptosis; shown by high caspase3/7 activity, coupled with increase in pro-apoptotic LvCaspase3 and LvCytochrome C, and decrease in pro-survival LvBcl2, LvIAP1, and LvIAP2 mRNA expression in hemocytes. Finally, LvPim1 knockdown renders shrimps more susceptible to V. parahaemolyticus infection. Taken together, our present data strongly suggest that LvPim1 is involved in modulating shrimp resistance to pathogen infection, promote hemocytes survival, and therefore plays a role in shrimp immune response.

Comparative transcriptomic analysis of shrimp hemocytes in response to acute hepatopancreas necrosis disease (AHPND) causing Vibrio parahemolyticus infection.

The recent emergence of acute hepatopancreas necrosis disease (AHPND) in shrimps has posed a major challenge in the shrimp aquaculture industry. The Pir toxin proteins carried by some strains of Vibrio parahaemolyticus are believed to play essential roles in the pathogenesis of AHPND. However, few studies have so far explored how the host immune system responds to these bacteria. In this study, AHPND V. parahaemolyticus (with Pir) and non-AHPND V. parahaemolyticus (without Pir) were injected into two groups of shrimps, and the hemocytes collected for comparative transcriptomic analyses. A total of 1064 differentially expressed genes (DEGs) were identified, of which 910 were up-regulated and 154 were down-regulated. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that many DEGs were involved in a number of biological processes such as cellular process, metabolic process and single-organism process in the AHPND V. parahaemolyticus injected group than the non-AHPND V. parahaemolyticus injected group. Among these, major metabolic processes such as carbohydrate metabolism, lipid metabolism and amino acid metabolism were further identified as the major responsive gene groups. We observed that genes involved in cell growth and anti-apoptosis including src, iap2, cas2, cytochrome P450, gst and cytochromecoxidase were strongly activated in the AHPND V. parahaemolyticus group than in the non-AHPND V. parahaemolyticus group. Collectively, our results unveiled that shrimp hemocytes respond to AHPND related strain of Vibrio parahaemolyticus infection at the transcriptional level, which is useful in furthering our understanding of AHPND.