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In vertebrate, MIP-T3 (microtubule-interacting protein associated with TRAF3) functions as a regulator of innate immune response that involves many cellular processes. However, the immune response regulated by shrimp (an arthropod) MIP-T3 remains unrevealed. In the present study, a MIP-T3 homolog from shrimp Litopenaeus vannamei (named as LvMIP-T3) was cloned and identified. LvMIP-T3 had a 2076 bp open reading frame (ORF), encoding a polypeptide of 691 amino acids that contained a classic coiled-coil domain in the C-terminal that showed a high degree of conservation to other homologs. LvMIP-T3 could interact with LvTRAF6, a member of the canonical NF-κB pathway, but not LvTRAF3, which implies that LvMIP-T3 is able to regulate NF-κB activity via its interaction with LvTRAF6. In addition, LvMIP-T3 was substantially inducted in response to white spot syndrome virus (WSSV) challenge, and we demonstrated that LvMIP-T3 facilitated the expression of NF-κB-mediated several Penaeidins (antimicrobial peptides, AMPs) to oppose infection. Taken together, we identified a MIP-T3 homolog from shrimp L. vannamei that played a positive role in the TRAF6/NF-κB/AMPs axis mediated defense response, which will contribute to better understand the regulator relationship among members of the canonical NF-κB pathway in shrimp, and provides some insights into disease resistance breeding. • LvMIP-T3 was response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. • LvMIP-T3 interacted with LvTRAF6 but not LvTRAF3. • LvMIP-T3 regulated the activity (phosphorylation) of LvDorsal and LvRelish. • LvMIP-T3 facilitated Penaeidins expression to protect shrimp from WSSV infection. [ABSTRACT FROM AUTHOR]

A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)–Vago–JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF–IFN–JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT–IFI6-16–caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069 , wsv056 , and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway. [ABSTRACT FROM AUTHOR]

Thioester-containing proteins (TEPs) are present in a wide range of species from deuterostomes to protostomes and are thought to be involved in innate immunity. In the current study, a TEP gene homologous to insect TEPs (iTEP) from the crustacean Litopenaeus vannamei , named LvTEP1, is cloned and functionally characterized. The open reading frame (ORF) of LvTEP1 is 4383 bp in length, encoding a polypeptide of 1460 amino acids with a calculated molecular weight of 161.1 kDa LvTEP1, which is most similar to other TEPs from insects, contains some conserved sequence features, including a N-terminal signal peptide, a canonical thioester (TE) motif, and a C-terminal distinctive cysteine signature. LvTEP1 is expressed in most immune-related tissues, such as intestine, epithelium, and hemocytes, and the mRNA level of LvTEP1 is upregulated in hemocytes after bacterial and viral challenges, indicating its involvement in the shrimp innate immune response. An expression assay in Drosophila S2 cells shows LvTEP1 to be a full-length secretory protein, and processed forms are present in the supernatant. Of note, only the processed form of LvTEP1 protein can bind to both the gram-negative bacterium Vibrio parahaemolyticus and the gram-positive bacterium Staphylococcus aureus in vitro , and its abundance can be induced after bacterial treatment. Moreover, knockdown of LvTEP1 renders shrimps more susceptible to both V. parahaemolyticus and S. aureus , as well as white spot syndrome virus (WSSV) infection, suggesting its essential defensive role against these invading microbes. We also observe that the expression of LvTEP1 is regulated in a manner dependent on both NF-κB and AP-1 transcription factors in naive shrimps and in vitro , suggesting that LvTEP1 could be poised in the body cavity prior to infection and thus play an important role in basal immunity. Taken together, our findings provide some in vitro and in vivo evidence for the involvement of LvTEP1 in shrimp innate immunity and provide some insight into its expression regulation mediated by multiple transcription factors or signaling pathways. [ABSTRACT FROM AUTHOR]

LvTAK1, a member of transforming growth factor β-activated kinase 1 (TAK1) families, has been identified from Litopenaeus vannamei in this study. The full length of LvTAK1 is 2670 bp, including a 2277 bp open reading frame (ORF) that encoded a putative protein of 758 amino acids with a calculated molecular weight of ∼83.4 kDa LvTAK1 expression was most abundant in muscles and was up-regulated in gills after LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I:C) and WSSV challenge. Both in vivo and in vitro experiments indicated that LvTAK1 could activate the expression of several antimicrobial peptide genes (AMPs). In addition, the dsRNA-mediated knockdown of LvTAK1 enhanced the susceptibility of shrimps to Vibrio parahaemolyticus, a kind of Gram-negative bacteria. These results suggested LvTAK1 played important roles in anti-bacterial infection. CoIP and subcellular localization assay demonstrated that LvTAK1 could interact with its binding protein LvTAB2, a key component of IMD pathway. Moreover, over-expression of LvTAK1 in Drosophila S2 cell could strongly induce the promoter activity of Diptericin (Dpt), a typical AMP which is used to read out of the activation of IMD pathway. These findings suggested that LvTAK1 could function as a component of IMD pathway. Interestingly, with the over-expression of LvTAK1 in S2 cell, the promoter activity of Metchnikowin (Mtk), a main target gene of Toll/Dif pathway, was up-regulated over 30 times, suggesting that LvTAK1 may also take part in signal transduction of the Toll pathway. In conclusion, we provided some evidences that the involvement of LvTAK1 in the regulation of both Toll and IMD pathways, as well as innate immune against bacterial infection in shrimp. [ABSTRACT FROM AUTHOR]

Ammonia nitrogen (ammonia-N) accumulation is pointed out as an environmental stress factor in the aquatic culture system, and has been shown to increase the susceptibility of aquatic organisms to infection. However, the mechanism by which ammonia-N stress induces a higher susceptibility to infection or disease outbreak remains largely unknown. Herein, we cloned and identified an interleukin-1 receptor homolog from the shrimp Litopenaeus vannamei (designated as LvIL-1R), and uncovered a LvIL-1R – LvRelish axis mediated antibacterial signaling that could induce the expression of antimicrobial peptides (AMPs) and thus conferred protection against Vibrio parahaemolyticus infection. Besides, we observed that shrimps became more susceptible to bacterial infection under ammonia-N stress, which attributed to that the LvIL-1R – LvRelish-AMPs antibacterial axis was restrained by the elevated ammonia-N concentration in hemolymph. Our results reveal how environmental (ammonia-N) stress increases shrimp susceptibility to infection, thereby providing some insights into the development of an effective environmental control strategy for disease prevention and treatment in aquaculture. [Display omitted] • A novel IL-1R–Relish AMPs axis was firstly established in shrimp. • Ammonia-N stress resulted in the elevated ammonia-N concentration in hemolymph. • Ammonia-N stress restrained LvIL-1R mediated antibacterial signaling. • Ammonia nitrogen stress increased shrimp susceptibility to bacterial infection. [ABSTRACT FROM AUTHOR]

p38 mitogen-actived protein kinases (MAPKs) broadly exist from yeast to mammals and participate in diverse cellular responses to various stimuli, whose activation can be induced by the MAPK kinase 6 (MKK6). In this study, a novel MKK6 homolog from Litopenaeus vannamei (LvMKK6) was cloned and characterized. The transcript of LvMKK6 was 1465 bp long with an open reading frame (ORF) of 987 bp that encoded a polypeptide of 328 amino acids. LvMKK6 was a both cytoplasmic- and nuclear-localized protein and its expression was up-regulated with the treatment of different stimuli including LPS, Vibrio parahaemolyticus , Staphylococcus aureus , Poly (I:C) and white spot syndrome virus (WSSV). Overexpression of LvMKK6 could lead to activate the promoter activities of several antimicrobial peptides (AMPs) such as PEN4. The further investigation demonstrated that LvMKK6 could interact with and phosphorylate Lvp38, suggesting LvMKK6 was an activator of Lvp38. Knockdown of LvMKK6 caused attenuate expression of several AMPs and resulted in the higher mortality of shrimp under V. parahaemolyticus infection, suggesting LvMKK6 could play vital roles in defense against bacterial infection. Interestingly, silencing of LvMKK6 led to the lower virus loads and suppressed viral gene (VP28) expression during WSSV challenge. In addition, overexpression of LvMKK6 promoted the promoter activities of 19 WSSV immediate-early genes such as wsv069, wsv249, wsv108 and wsv403. These results suggested that LvMKK6 could be used by WSSV. Above all, these data provided experimental evidences that participation of LvMKK6 in regulating AMPs and host defense against bacteria, as well as the immune response to WSSV infection. [ABSTRACT FROM AUTHOR]

In Drosophila , TAB2, an important intermediate in the IMD signaling pathway, plays critical roles in the innate immune response in response to bacterial and viral infection. However, the role of TAB-related proteins in the immune response of shrimp has not yet been established. Here, we reported the identification of a TAB2-like gene in Litopenaeus vannamei designated as LvTAB2. The full-length cDNA of LvTAB2 was 2160 bp with an open reading frame of 1827 bp, which encoded a putative protein of 608 amino acids including a ubiquitin binding domain (CUE) at the N-terminal and a Zinc Finger domain (ZnF) at the C-terminus. Real-time RT-PCR analysis showed that LvTAB2 was expressed in all tested tissues and the expression levels of LvTAB2 in gills and hemocytes were positively induced in response to LPS, Vibrio parahemolyticus and White Spot Syndrome Virus (WSSV) challenges. Dual luciferase reporter assays demonstrated that LvTAB2 was able to induce the expression of antimicrobial peptide (AMP) genes, including Drosophila Attacin A and shrimp Penaeidins. Interestingly, over-expression of LvTAB2 could up-regulate the promoter activities of L. vannamei Vago1, Vago3 and Vago4 genes in S2 cells. To our knowledge, it was the first report that TAB2 participated in innate immune signaling to regulate the expression of Vago genes in invertebrates. Moreover, RNAi-mediated knockdown of LvTAB2 enhanced sensitivity of L. vannamei to Vibrio parahaemolyticus infection and caused elevated virus loads after WSSV infection. We suggested that the LvTAB2 may play important roles in the shrimp innate immunity. [ABSTRACT FROM AUTHOR]

Growing evidence indicates that activator protein-1 (AP-1) plays a major role in stimulating the transcription of immune effector molecules in cellular response to an incredible array of stimuli, including growth factors, cytokines, cellular stresses and bacterial and viral infection. Here, we reported the isolation and characterization of a cDNA from Litopenaeus vannamei encoding the full-length c-Fos protein (named as Lvc-Fos). The predicted amino acid sequences of Lvc-Fos contained a basic-leucine zipper (bZIP) domain, which was characteristic of members of the AP-1 family. Immunoprecipitation and native-PAGE assays determined that Lvc-Fos could interact with the Lvc-Jun, a homolog of c-Jun family in L. vannamei , in a heterodimer manner. Further investigation demonstrated that Lvc-Fos and Lvc-Jun were expressed in all tested tissues and located in the nucleus. Real-time RT-PCR analysis showed both Lvc-Fos and Lvc-Jun in gills were up-regulated during Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges. In addition, reporter gene assays indicated Lvc-Fos and Lvc-Jun could activate the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp, as well as WSSV immediate early (IE) genes wsv069 and wsv249 , in a different manner. Knockdown of Lvc-Fos or Lvc-Jun by RNA interference (RNAi) resulted in higher mortalities of L. vannamei after infection with V. parahaemolyticus , suggesting that Lvc-Fos and Lvc-Jun might play protective roles in bacterial infection. However, silencing of Lvc-Fos or Lvc-Jun in shrimp caused lower mortalities and virus loads under WSSV infection, suggesting that Lvc-Fos and Lvc-Jun could be engaged for WSSV replication and pathogenesis. In conclusion, our results provided experimental evidence and novel insight into the roles of L. vannamei AP-1 in bacterial and viral infection. [ABSTRACT FROM AUTHOR]

Hemolin is a specific immune protein belonging to immunoglobulin superfamily and firstly identified in insects. Growing evidences suggest that Hemolin can be activated by bacterial and viral infections and may play an important role in antimicrobial immunity. In this paper, we firstly identified a Hemolin-like protein from Litopenaeus vannamei (LvHemolin). Sequence analysis showed that LvHemolin shares high similarity with insect Hemolins and is mainly composed of seven immunoglobulin (Ig) domains which form a ‘horseshoe’ tertiary structure. Tissue distribution analysis demonstrated that LvHemolin mainly expressed in stomach, gill, epithelium and pyloric cecum of L. vannamei . After challenge with pathogens or stimulants, expression of LvHemolin was significantly up-regulated in both gill and stomach. Agglutination analysis demonstrated that recombinant LvHemolin protein purified from Escherichia coli could accelerate the agglutination of Vibrio parahaemolyticus , E. coli , Staphylococcus aureus , and Bacillus subtilis in the presence of Ca 2+ . To verify the immune function of LvHemolin in vivo , shrimps were injected with gene-specific dsRNA, followed by challenge with white spot syndrome virus (WSSV) or V . parahaemolyticus . The results revealed that silence of LvHemolin could increase the cumulative mortalities of shrimps challenged by pathogens and increase the WSSV copies in shrimp tissues. These suggested that Hemolin could play an important role in shrimp innate immune defense against bacterial and viral infections. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) play multiple roles in innate immune defense against invading pathogens in both vertebrates and invertebrates. In this study, a new C-type lectin gene from pacific white shrimp Litopenaeus vannamei (designated as LvCTL4 ) was cloned by rapid amplification of the cDNA ends (RACE) method. The full-length cDNA of LvCTL4 was 563 bp with open reading frame (ORF) of 471 bp encoding a polypeptide of 156 amino acids, including a putative signal sequence and a single C-type lectin-like domain (CTLD). The CTLD of 137 amino acid residues contained a mutated ‘EPA’ (Glu 121 -Pro 122 -Ala 123 ) motif in the calcium-binding site 2 and three conserved disulfide bonds involved in structure maintenance. Tissue expression analysis showed LvCTL4 was ubiquitously distributed with high levels in gill, intestine, epithelium and hepatopancreas. The expression of LvCTL4 in gill was up-regulated in response to Vibrio parahaemolyticus challenge. RNAi knock-down of the LvCTL4 gene significantly increased mortality after V. parahaemolyticus infection. A 103 bp 5′ flanking promoter sequence was obtained using the genome walking method and it contained a conserved NF-κB binding motif. Dual-Luciferase assay showed both LvDorsal and LvRelish could up regulate the promoter activity of LvCTL4 . This is the first report that a shrimp C-type lectin can be regulated by both LvDorsal and LvRelish . These findings provided novel insights into the regulation of shrimp CTLs expression. [ABSTRACT FROM AUTHOR]

MAPKKK15 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, which plays an important role in multiple biological progresses triggered by cellular stresses. In this study, a new homolog of MAPKKK15 has been cloned and identified from shrimp Litopenaeus vannamei (named as LvMAPKKK15). The full length of LvMAPKKK15 transcript was 4597 bp, including a 4317 bp open reading frame (ORF) that encoded a putative protein of 1438 amino acids. LvMAPKKK15 was abundantly expressed at early development stages with the highest transcriptional level in P12 stage. In adult shrimp, tissue distribution showed that LvMAPKKK15 was highly expressed in eyestalk, nerve and hemocytes. Besides, the induced transcription of LvMAPKKK15 could respond to the challenges of Poly (I: C) and white spot syndrome virus (WSSV). RNAi-mediated knockdown of LvMAPKKK15 caused shrimp less sensitive to WSSV infection, manifested by lessened virus loads, higher survival rate and higher transcription levels of anti-viral factors LvVagos. The results were further supported by the dual-luciferase reporter assays that the LvMAPKKK15 was able to up-regulate the promoter activities of several WSSV immediate early (IE) genes, but suppress the promoter activities of LvVago1–5 in vitro of cells. Together, these results suggested that LvMAPKKK15 was transcribed in larva development stages and played a role during WSSV pathogenesis. • A new MAPKKK15 homolog was identified from shrimp Litopenaeus vannamei. • The structural domain and subcellular location of LvMAPKKK15 was identified. • LvMAPKKK15 was ubiquitously expressed in all larva stage with high expression in postlarva stage. • The expressional levels of LvMAPKKK15 were up-regulated after WSSV and Poly (I: C) challenges. • LvMAPKKK15 played an important role during WSSV pathogenesis. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are the most important players in the innate immune system, providing a principal first-line of defense against the invading pathogens. Crustin, a type of whey acidic protein (WAP) domain-containing and cationic cysteine-rich AMP, can function in a protease inhibition or an effector molecule manner. In the present study, a new Crustin was cloned and identified from Pacific white shrimp Litopenaeus vannamei and designated as LvCrustinA. The full-length cDNA of LvCrustinA was 687 bp, with a 519 bp open reading frame (ORF) that encoded a peptide of 172 amino acids. Domain analysis indicated that LvCrustinA contained a Glycine-rich region in the N-terminal and a single WAP domain within eight cysteines in the C-terminal. The 5′ upstream regulatory sequence of 1249 bp (promoter) was obtained using a genome walking method, and it contained several conserved transcription factors binding motifs including NF-κB, AP-1 and STAT (Signal transducers and activators of transcription). Dual-reporter assay showed that NF-κB transcription factors LvDorsal and LvRelish, and AP-1 transcription factor Lvc-Jun could up-regulate the promoter activity of LvCrustinA, suggesting that NF-κB and JNK-c-Jun pathways could be involved in regulating the expression of LvCrustinA. Moreover, LvCrustinA was abundantly expressed in immune related tissues such as gill, hemocyte and epithelium, and its expression was up-regulated in response to Vibrio parahaemolyticus and White spot syndrome virus (WSSV) challenges in gill tissue, suggesting that LvCrustinA could be involved in the host defense against bacterial and viral infection. Additionally, RNAi mediated knockdown of LvCrustinA resulted in shrimps with the higher cumulative mortality during V. parahaemolyticus and WSSV infection. Taken together, these results provided some insight into the expression and transcriptional regulatory role of LvCrustinA, and its defensive role against pathogenic infection. [ABSTRACT FROM AUTHOR]

LvMKK4, a homologue of the mammalian mitogen-activated protein kinase kinase 4 (MKK4), was isolated and identified from Litopenaeus vannamei in the present study. The full-length cDNA of LvMKK4 is 1947 bp long, with an open reading frame (ORF) of 1185 bp encoding a putative protein of 388 amino acids. LvMKK4 contains several characteristic domains such as D domain, SIAKT motif and kinase domain, all of which are conserved in MAP kinase kinase family. Like mammalian MKK4 but not Drosophila MKK4, LvMKK4 could bind to, phosphorylate and activate p38 MAPK, which provided some insights into the signal transduction mechanism of MKK4-p38 cascade in invertebrates. Our real-time PCR data indicated that LvMKK4 was ubiquitously expressed in all tested tissues and extraordinarily abundant in muscle. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvMKK4 activated the transcription of antimicrobial peptide genes (AMPs), including Drosophila Attacin A, Drosomycin, and shrimp Penaeidins. Additionally, LvMKK4 was up-regulated in both intestine and hepatopancreas by a variety of inflammatory stimuli including LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I: C) and white spot syndrome virus. Furthermore, RNAi-mediated knockdown of LvMKK4 enhanced the sensitivity of L. vannamei to V. parahaemolyticus infection. These findings suggested that LvMKK4 played an important role in anti-bacterial response and could be a potential target for inflammation treatment. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase kinase 7 (MKK7) is a key signal transduction regulator in c-Jun N-terminal kinase (JNK) signaling pathway, which is involved in a wide range of physiological and pathological processes. In this study, we described the molecular cloning of a new member of MKK7 group from Litopenaeus vannamei named as LvMKK7. The full-length cDNA of LvMKK7 was 3093 bp in length, with an open reading frame (ORF) of 1440 bp encoding a putative protein of 479 amino acids. LvMKK7 contained a conserved kinase domain of 261 amino acids in which there was a characteristic S-K-A-K-T motif as a potential target site of phosphorylation by MKKK. Moreover, subcellular localization showed LvMKK7 was located in both the cytoplasm and the nucleus of Drosophila S2 cells. Real-time PCR indicated that LvMKK7 was universally expressed in all tested tissues and its expression in hepatopancreas was responsive to the challenge of LPS, Poly (I:C), Vibrio parahaemolyticus , Staphhylococcus aureus and white spot syndrome virus (WSSV). In addition, co-immunoprecipitation assay demonstrated that LvJNK was phosphorylated and activated by LvMKK7, which suggested LvMKK7 was the upper regulator of LvJNK. Furthermore, RNAi-mediated knockdown of LvMKK7 enhanced the sensitivity of shrimps to V. parahaemolyticus infection. Overall, our results suggested that LvMKK7 may play important roles in the shrimp innate immunity. [ABSTRACT FROM AUTHOR]

Highlights: [•] The first cloned and studied Pellino homolog gene (LvPellino) in Class Crustacea. [•] LvPellino functions as a positive regulator in the toll-like receptor pathway. [•] LvPellino may play a complex role in antiviral mechanisms and WSSV pathogenesis. [Copyright &y& Elsevier]

Phenylalanine hydroxylase (PAH) is involved in immune defence reactions by providing the starting material, tyrosine, to synthesise catecholamines and melanin. PAH is an important metabolic enzyme of aromatic amino acids and the rate-limiting enzyme in the hydroxylation of amino acid phenylalanine to tyrosine. In the present study, a PAH gene, LvPAH, was cloned and identified from Litopenaeus vannamei. The open reading frame (ORF) of LvPAH was 1383 bp, encoding a protein of 460 amino acids comprised of an ACT domain and a Biopterin_H domain. LvPAH was constitutively expressed in healthy L. vannamei , with the highest expression levels in the eyestalk and the lowest in the hepatopancreas. Both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus infection upregulated LvPAH expression in hemocytes, hepatopancreas and gills of L. vannamei. Inhibition of LvPAH resulted in a significantly lower survival rate of L. vannamei after WSSV infection than the control group, consistent with the observation that WSSV viral load was significantly higher in LvPAH-silenced L. vannamei. After a V. parahaemolyticus challenge, there was no significant difference between the survival rate of LvPAH-silenced and the control L. vannamei. However, the load of V. parahaemolyticus in LvPAH-silenced L. vannamei was significantly higher than the control population for L. vannamei. The effect of LvPAH on L. vannamei from a neuroendocrinological perspective was assessed by measuring l -DOPA, dopamine (DA) and noradrenaline (NE) levels in the hemocytes after the knockdown of LvPAH. The results showed that phenoloxidase (PO), l -DOPA and DA levels in the hemolymph of LvPAH-silenced L. vannamei were significantly decreased starting from 24hpi. In contrast, the NE levels in the hemolymph of shrimp decreased significantly at first and then increased. The results suggest that LvPAH may play an important role in antiviral and bacterial immunity in L. vannamei. • PAH gene was cloned from Litopenaeus vannamei. • LvPAH expression could be induced by WSSV and Vibrio parahaemolyticus infection. • dsRNA-LvPAH injection significantly decreased the survival rates of L. vannamei after WSSV infection. • The loads of WSSV and V. parahaemolyticus in dsRNA-LvPAH injected L. vannamei were significant higher than the control. • The melanisation was regulated by LvPAH silence. [ABSTRACT FROM AUTHOR]

Mammalian TAB1 has been previously identified as transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) binding protein, which functions as the activator of TAK1 and p38. This report, for the first time, identified and characterized the homolog of TAB1 in shrimp, to be specific, the homolog gene from Litopenaeus vannamei, containing a 1560-bp open reading frame (ORF) that encoded a putative protein of 519 amino acids with the conserved PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain in N-terminal and a TAK1 binding motif in C-terminus, has been cloned and named LvTAB1. LvTAB1 was most abundant in gills and its expression could respond significantly to a series of stimuli, including LPS, Vibrio parahemolyticus and Staphylococcus aureus . Moreover, Co-immunoprecipitation (Co-IP) experiments showed that LvTAB1 could combine with LvTAK1 as well as Lvp38, two members of IMD-NF-κB/MAPK pathway, which meant LvTAB1 could have a role in regulating the activities of these kinases. Over-expression of LvTAB1 in drosophila S2 cells could improve the transcriptional levels of antimicrobial peptide genes (AMPs) such as Diptericin (Dpt), the hallmark of drosophila NF-κB activated genes, indicating its activation effect on NF-κB pathway. Furthermore, suppression of LvTAB1 expression in vivo by RNA-interference increased the sensibility of shrimps to V. parahaemolyticus infection, implying its protective role against bacterial infection. In conclusion, these results provide some insight into the function of LvTAB1 during bacterial infection. [ABSTRACT FROM AUTHOR]

JAK/STAT pathway is one of cytokine signaling pathways and mediates diversity immune responses to protect host from viral infection. In this study, LvSOCS2, a member of suppressor of cytokine signaling (SOCS) families, has been cloned and identified from Litopenaeus vannamei . The full length of LvSOCS2 is 1601 bp, including an 1194 bp open reading frame (ORF) coding for a putative protein of 397 amino acids with a calculated molecular weight of ∼42.3 kDa. LvSOCS2 expression was most abundant in gills and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly (I: C) and white spot syndrome virus (WSSV). There are several STAT binding motifs presented in the proximal promoter region of LvSOCS2 and its expression was induced by LvJAK or LvSTAT protein in a dose dependent manner, suggesting LvSOCS2 could be the transcriptional target gene of JAK/STAT pathway. Moreover, the transcription of DmVir-1, a read out of the activation of JAK/STAT pathway in Drosophila , was promoted by LvJAK but inhibited by LvSOCS2, indicating that LvSOCS2 could be a negative regulator in this pathway and thus can form a negative feedback loop. Our previous study indicated that shrimp JAK/STAT pathway played a positive role against WSSV. In this study, RNAi-mediated knockdown of LvSOCS2 shrimps showed lower susceptibility to WSSV infection and caused lessened virus loads, which further demonstrated that the JAK/STAT pathway could function as an anti-viral immunity in shrimp. [ABSTRACT FROM AUTHOR]

Wnt signaling is known to control multiple of cellular processes such as cell differentiation, communication, apoptosis and proliferation, and is also reported to play a role during microbial infection. β-catenin is a key regulator of the Wnt signaling cascade. In the present study, we cloned and identified a β-catenin homologue from Litopenaeus vannamei termed Lvβ-catenin. The full-length of Lvβ-catenin transcript was 2797 bp in length within a 2451 bp open reading frame (ORF) that encoded a protein of 816 amino acids. Lvβ-catenin protein was comprised of several characteristic domains such as an N-terminal region of GSK-β consensus phosphorylation site and Coed coil section, a central region of 12 continuous Armadillo/β-Catenin-like repeat (ARM) domains and a C-terminal region. Real-time PCR showed Lvβ-catenin expression was responsive to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infection. Dual-reporter analysis showed that over-expression of Lvβ-catenin could induce activation of the promoter activities of several antimicrobial peptides (AMPs) such as shrimp PEN4, suggesting that Lvβ-catenin could play a role in regulating the production of AMPs. Knockdown of Lvβ-catenin enhanced the sensitivity of shrimps to V. parahaemolyticus and WSSV challenge, suggesting Lvβ-catenin could play a positive role against bacterial and viral pathogens. In summary, the results presented in this study provided some insights into the function of Wnt/β-catenin of shrimp in regulating AMPs and the host defense against invading pathogens. [ABSTRACT FROM AUTHOR]

Death domain-associated factor 6 (Daxx) is a Fas-binding protein that mediates the activation of Jun amino-terminal kinase (JNK) pathway and Fas-induced apoptosis. In this study, a crustacean Daxx ( LvDaxx ) was firstly cloned and identified from Pacific white shrimp Litopenaeus vannamei . The LvDaxx cDNA was 2644 bp in length with an Open Reading Frame (ORF) of 2217 bp. Sequence analysis indicated that LvDaxx contained a single Daxx domain and two nuclear localization signals (NLSs) and shared a similarity with Drosophila melanogaster Daxx. LvDaxx was a nuclear-localized protein that was expressed highest in hemocytes and could be up-regulated in pathogen- and stimulant-challenge shrimps. LvDaxx could activate the artificial promoter containing an NF-κB binding site and the promoters of white spot syndrome virus (WSSV) ie1 gene and arthropod antimicrobial peptides (AMPs), suggesting LvDaxx could be involved in the activation of the NF-κB pathway. Knock-down of LvDaxx in vivo resulted in down-regulation of shrimp AMPs and reduction of WSSV copies in tissues. Furthermore, suppression of LvDaxx significantly decreased the mortality of WSSV-infected shrimps, but increased the mortality of Vibrio Parahaemolyticus -infected shrimps. Thus, these suggested that LvDaxx could play a role in the innate immunity against Vibrio parahaemolyticus in L. vannamei , while in the antiviral response, LvDaxx may be hijacked by WSSV and play a complex role in WSSV pathogenesis. [ABSTRACT FROM AUTHOR]

The JAK/STAT signaling pathways are conserved in evolution and mediate diversity immune responses to virus infection. In the present study, a Janus kinase (designated as LvJAK) gene was cloned and characterized from Litopenaeus vannamei . LvJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7 and showed 19% identity (34% similarity) and 21% identity (35% similarity) to Drosophila Hopscotch protein and Human JAK2 protein, respectively. The mRNA of LvJAK was highly expressed in hepatopancreas of L. vannamei and its expression level was prominently upregulated after the stimulation of Poly (I:C) and white spot syndrome virus (WSSV) challenges. There were 10 putative STAT binding motifs in the promoter region of LvJAK, and it could be regulated by LvJAK self or (and) LvSTAT, suggesting that LvJAK is the JAK/STAT pathway target gene and could function as a positive regulator to form a positive feedback loop. In addition, the silencing of LvJAK caused higher mortality rate and virus load, suggesting that LvJAK could play an important role in defense against WSSV. This is the first report about the complete set of JAK/STAT proteins in shrimp and the results provide the evidence of the positive feedback loop mediated by JAK protein present in the JAK/STAT pathway in invertebrates. [ABSTRACT FROM AUTHOR]

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway was known to participate in dozens of immune responses in organisms. Domeless, first identified in Drosophila melanogaster , is a unique receptor involved in invertebrate JAK/STAT pathway. In this study, a cytokine receptor ( LvDOME ) was identified in Litopenaeus vannamei . The LvDOME cDNA was 5178 bp in length with an Open Reading Frame (ORF) of 4191 bp. LvDOME contained two cytokine binding modules (CBMs) and three fibronectin-type-III-like (FNIII) domains, similar to most vertebrate IL-6 receptors. LvDOME was expressed highest in shrimp muscle and could be up-regulated in the late stage of white spot syndrome virus (WSSV) infection. LvDOME could significantly enhance the activity of the WSSV wsv069 gene promoter through acting on the STAT-binding motif, suggesting LvDOME could activate the JAK/STAT pathway. Moreover, knockdown of LvDOME resulted in lower cumulative mortality of shrimps and less WSSV copies, suggesting LvDOME may be hijacked by WSSV to benefit virus replication. To our knowledge, this is the first report on the receptor of JAK/STAT pathway in shrimp. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated ‘EPD’ motif in Ca 2+ -binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5′flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-luciferase reporter assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could be regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but cannot agglutinate Gram-positive bacteria Streptococcus agalactiae . The agglutination activity of LvCTL3 was abolished when Ca 2+ was chelated with EDTA, suggesting the function of LvCTL3 is Ca 2+ -dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] The previously reported Litopenaeus vannamei Pelle is really a Tube. [•] A real L. vannamei Pelle was identified. [•] Isoforms of Tube are present in invertebrates. [•] Isoforms of L. vannamei Tube play different roles during immune responses. [•] Signal transduction of invertebrate TLR pathway has various regulatory patterns. [Copyright &y& Elsevier]