Shrimp Innate Immunity Research Articles

Identification and function of penaeidin 3 and penaeidin 5 in Fenneropenaeus merguiensis

Antimicrobial peptides (AMPs) participate in immune defenses of invertebrate, vertebrate and plant species. As a kind of AMPs, penaeidins play important roles in innate immunity of shrimp. In this study, two penaeidin homologues termed FmPEN3 and FmPEN5 were cloned and identified from Fenneropenaeus merguiensis for the first time. The complete open reading frames (ORFs) of FmPEN3 and FmPEN5 were 216 bp and 240 bp, encoding 71 and 79 amino acids, respectively. Both FmPEN3 and FmPEN5 contain an N-terminal proline-rich domain (PRD) and a C-terminal cysteine-rich domain (CRD). The genome structure of FmPEN3 and FmPEN5 genes both consist of 2 exons and 1 intron. qPCR analysis showed that FmPEN3 was constitutively expressed but FmPEN5 transcripts were found only in hemocytes, gills, epidermis, nerve and pyloric cecum. The FmPEN3 and FmPEN5 expression were responsive to Vibrio parahaemolyticus and Micrococcus lysodeikticus infection and their transcription levels were downregulated by RNAi silencing of the transcription factors FmDorsal and FmRelish. In addition, recombinant proteins of FmPEN3 (rFmPEN3) and FmPEN5 (rFmPEN5) were successfully expressed in E. coli. The antibacterial assays revealed that rFmPEN3 and rFmPEN5 could inhibit the growth of M. lysodeikticus but only rFmPEN5 could inhibit the growth of V. parahaemolyticus in vitro. In summary, the results presented in this study indicated the functions of FmPEN3 and FmPEN5 played in anti-bacterial immunity of F. merguiensis, providing some insights into the function of AMPs in shrimp.

Effects of Bacillus aryabhattai TBRC8450 on vibriosis resistance and immune enhancement in Pacific white shrimp, Litopenaeus vannamei

The use of probiotics in aquaculture is a practical alternative to promote animal health and disease prevention. Meanwhile, this practice can also reduce the use of prophylactic antibiotics. The purpose of this study was to identify candidate probiotics that could control pathogen populations in host's gastrointestinal (GI) tract and stimulate host immunity in shrimp aquaculture. Bacillus aryabhattai TBRC8450, a bacterial strain isolated from the environment in a shrimp farm, has an antimicrobial activity against many pathogenic strains of Vibrio harveyi and V. parahaemolyticus. Supplementation of B. aryabhattai to Pacific white shrimp (Litopenaeus vannamei) not only decreased the abundance of Vibrio populations, but also shifted the bacterial community in the shrimp GI tract. We found that supplementation of B. aryabhattai triggered shrimp innate immunity and antioxidant activities. mRNA expression of genes encoding microbial peptides and antioxidant enzymes, including C-type lectin, penaeidin-3, heat shock protein 60, thioredoxin, and ferritin, was significantly upregulated in the hepatopancreas of shrimp fed B. aryabhattai. Furthermore, phenoloxidase activity in the hemocytes and the total antioxidant activity in the plasma were increased, indicating enhanced immune and antioxidant responses at the systemic level. In contrast, supplementation of B. aryabhattai had no effect on the total hemocyte count and superoxide dismutase activity in the plasma and hepatopancreas. Importantly, a pathogen challenge test using V. harveyi 1562 showed a significant increase in survival rates of shrimp fed B. aryabhattai compared to the control group. Our findings suggest that B. aryabhattai TBRC8450 can likely be used as a probiotic to reduce the population of V. harveyi in the shrimp GI tract and to enhance shrimp innate immunity and antioxidant capacity for vibriosis resistance in shrimp aquaculture.

France: Carbios/Carbiolice – masterbatch from recycled plastic

Hemocyanin is a copper containing respiratory glycoprotein in arthropods and mollusks, which also have multiple functions in vivo. Recent studies have shown that hemocyanin could generate several peptides, which play important roles in shrimp innate immunity. However, how these hemocyanin-derived peptides are generated is still largely unknown. In this study, we report for the first time that the generation of hemocyanin-derived peptides was closely correlated with trypsin expression in shrimp hepatopancreas following infection with different bacteria. RNA interference (RNAi) mediated knockdown of trypsin or treatment with the serine protease inhibitor, aprotinin, resulted in significant decrease in the levels of hemocyanin-derived peptides. Moreover, recombinant trypsin (rTrypsin) was able to hydrolyse hemocynin in vitro with the hydrolysate having a high bacterial agglutination activity while the denatured hemocynin untreated with rTrypsin lost its agglutination activity. Taken together, our current results showed that the generation of hemocyanin-derived peptides correlates with an increase trypsin expression.

Effects of environmental stress on shrimp innate immunity and white spot syndrome virus infection

The shrimp aquaculture industry is plagued by disease. Due to the lack of deep understanding of the relationship between innate immune mechanism and environmental adaptation mechanism, it is difficult to prevent and control the diseases of shrimp. The shrimp innate immune system has received much recent attention, and the functions of the humoral immune response and the cellular immune response have been preliminarily characterized. The role of environmental stress in shrimp disease has also been investigated recently, attempting to clarify the interactions among the innate immune response, the environmental stress response, and disease. Both the innate immune response and the environmental stress response have a complex relationship with shrimp diseases. Although these systems are important safeguards, allowing shrimp to adapt to adverse environments and resist infection, some pathogens, such as white spot syndrome virus, hijack these host systems. As shrimp lack an adaptive immune system, immunization therapy cannot be used to prevent and control shrimp disease. However, shrimp diseases can be controlled using ecological techniques. These techniques, which are based on the innate immune response and the environmental stress response, significantly reduce the impact of shrimp diseases. The object of this review is to summarize the recent research on shrimp environmental adaptation mechanisms, innate immune response mechanisms, and the relationship between these systems. We also suggest some directions for future research.

Antimicrobial activity of a serine proteinase inhibitor SPIPm5 from the black tiger shrimp Penaeus monodon

A two-domain Kazal-type serine proteinase inhibitor, SPIPm5, from Penaeus monodon was studied. Its transcript was expressed in all tissues tested including the hemocytes, stomach, gill, lymphoid organ, muscle, intestine and heart albeit less in hepatopancreas and eyestalk. The expression of SPIPm5 gene was also up-regulated by heat stress, white spot syndrome virus (WSSV) infection and yellow head virus (YHV) infection. Injection of recombinant rSPIPm5 protein into normal shrimp to mimic heat stress condition did not have or had little stimulating effect on the expression of other immune genes: crustinPm1, penaeidin3, penaeidin5, Hsp70, SPIPm2 and SPIPm5. Like some other proteinase inhibitors, the rSPIPm5 could inhibit the hemolymph proPO activity. In survival experiments, the rSPIPm5 could prolong the life of WSSV-infected shrimp similar to the effect of heat stress. The rSPIPm5 also helped the YHV-, Vibrio harveyi- and V. parahaemolyticus-infected shrimp survive longer. The increased endurance against microbial infection was due to the inhibitory effects presumably activated by rSPIPm5 on viral replication and bacterial growth but not the expression of antimicrobial peptides. Therefore, the SPIPm5 plays an important role in shrimp innate immunity against the viral and bacterial infection.

Endogenous molecules released by haemocytes receiving Sargassum oligocystum extract lead to downstream activation and synergize innate immunity in white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei haemocytes receiving immunostimulating Sargassum oligocystum extract (SE) caused necrosis in haemocyte cells, which released endogenous EM-SE molecules. This study examined the immune response of white shrimp L. vannamei receiving SE and EM-SE in vitro and in vivo. Shrimp haemocytes receiving SE exhibited degranulation, changes in cell size and cell viability, necrosis and a release of EM-SE. Shrimp haemocytes receiving SE, EM-SE, and the SE + EM-SE mixture (SE + EM-SE) increased their phenoloxidase (PO) activity which was significantly higher in shrimp haemocytes receiving the SE + EM-SE mixture. Furthermore, shrimp haemocytes receiving EM-SE showed degranulation and changes in cell size and cell viability. Shrimp receiving SE, EM-SE, and SE + EM-SE all increased their immune parameters, phagocytic activity, clearance efficiency and resistance to Vibrio alginolyticus, being significantly higher in shrimp receiving SE + EM-SE. Meanwhile, the recombinant lipopolysaccharide- and β-1,3-glucan binding protein of L. vannamei (rLvLGBP) was bound to SE, EM-SE, and SE + EM-SE. We conclude that in shrimp haemocytes receiving a non-self molecule, SE in dying cells released EM-SE which led to downstream activation and synergization of the immune response. This study demonstrated that the innate immunity of shrimp was elicited and enhanced by a mixture of endogenous molecules and exogenous substances (or immunostimulants).

Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection

This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P < .01) in the hemocytes of shrimp, while myosin was significantly up-regulated (P < .01). We demonstrated that mjERT is very important for the progression of WSSV infection and that the cumulative mortality of WSSV-infected and V. alginolyticus-infected shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC.

Trypsin of Litopenaeus vannamei is required for the generation of hemocyanin-derived peptides

Hemocyanin is a copper containing respiratory glycoprotein in arthropods and mollusks, which also have multiple functions in vivo. Recent studies have shown that hemocyanin could generate several peptides, which play important roles in shrimp innate immunity. However, how these hemocyanin-derived peptides are generated is still largely unknown. In this study, we report for the first time that the generation of hemocyanin-derived peptides was closely correlated with trypsin expression in shrimp hepatopancreas following infection with different bacteria. RNA interference (RNAi) mediated knockdown of trypsin or treatment with the serine protease inhibitor, aprotinin, resulted in significant decrease in the levels of hemocyanin-derived peptides. Moreover, recombinant trypsin (rTrypsin) was able to hydrolyse hemocynin in vitro with the hydrolysate having a high bacterial agglutination activity while the denatured hemocynin untreated with rTrypsin lost its agglutination activity. Taken together, our current results showed that the generation of hemocyanin-derived peptides correlates with an increase trypsin expression.

A mannose-specific C-type lectin from Fenneropenaeus merguiensis exhibited antimicrobial activity to mediate shrimp innate immunity

Being one type of pattern recognition receptors (PRRs), lectins exhibit a crucial role in the defense mechanism of invertebrates which are deficient in an adaptive immune system. A new C-type lectin called FmLC3 was isolated from hepatopancreas of Fenneropenaeus merguiensis by cloning approaches, RT-PCR and 5′ and 3′ RACE (rapid amplification of cDNA ends). A full-length cDNA of FmLC3 contains 607 bp with one open reading frame of 480bp, encoding a 159-amino acids peptide. The predicted primary structure of FmLC3 is composed of a signal peptide, a carbohydrate recognition domain with an EPN motif and one Ca2+ binding site-2, including a double-loop region assisted by two conserved disulfide linkages. FmLC3 had a molecular mass of 17.96kDa and pI of 4.92. In normal or unchallenged shrimp, the mRNA expression of FmLC3 was detected only in hepatopancreas whilst its native proteins were found in hemolymph, heart, stomach and intestine but not in the expressed tissue, indicating that after being synthesized in hepatopancreas, FmLC3 would be secreted to other tissues. The significant up-regulation of FmLC3 was manifested in shrimp challenged with Vibrio harveyi or white spot syndrome virus. After knockdown with gene-specific double-stranded RNA and following by co-pathogenic inoculation, the FmLC3 expression was severely suppressed with coherence of increasing in cumulative mortality and reduction of the median lethal time. Recombinant FmLC3 (rFmLC3) had agglutinating activity towards diverse bacterial strains in a Ca2+-dependent manner. Its activity was inhibited by lipopolysaccharide and mannose, implying that FmLC3 was mannose-binding C-type lectin. Moreover, rFmLC3 could bind directly to various microbial strains with Ca2+-requirement. Otherwise, rFmLC3 exhibited the antimicrobial activity by inhibiting effectively the microbial growth in vitro. All these results signified that FmLC3 might act as PRR to recognize with a broad specificity for diverse pathogens, and contribute in shrimp immune response via the agglutination, binding and antimicrobial activity.

Lipopolysaccharide-specific binding C-type lectin with one CRD domain from Fenneropenaeus merguiensis (FmLC4) functions as a pattern recognition receptor in shrimp innate immunity

In crustaceans, an innate immune system is solely required because they lack an adaptive immunity. One kind of pattern recognition receptors (PRRs) that plays a particular role in the innate immunity of aquatic shrimp is lectin. A new diverse C-type lectin (FmLC4) was cloned from the hepatopancreas of Fenneropenaeus merguiensis by using RT-PCR and 5′ and 3′ rapid amplification of cDNA ends approaches. A full-length FmLC4 cDNA comprises 706 bp with an open reading frame of 552 bp, encoding a peptide of 184 amino acids. The predicted primary sequence of FmLC4 consists of a signal peptide of 19 amino acids, a molecular mass of 20.4 kDa, an isoelectric point of 5.13, one carbohydrate recognition domain with a QPD motif and a Ca2+ binding site as well as a double-loop characteristic supported by two conserved disulfide bonds. The FmLC4 mRNA expression was found only in the hepatopancreas of normal shrimp and significantly up-regulated upon challenge the shrimp with Vibrio harveyi or white spot syndrome virus (WSSV). Recombinant FmLC4 (rFmLC4) could agglutinate various bacterial strains with Ca2+-dependence. Lipopolysaccharide (LPS) could specifically inhibit the agglutinating activity and potently bind to rFmLC4, indicating that FmLC4 was LPS-specific binding C-type lectin. Moreover, rFmLC4 itself displayed the in vivo effective clearance of the pathogenic bacterium V. harveyi. Altogether, FmLC4 may serve as LPS-specific PRR to recognize opportunistic bacterial and viral pathogens, and thus to play a role in the immune defense of aquatic shrimp via the binding and agglutination.

The crustin-like peptide plays opposite role in shrimp immune response to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection

Crustin is an antimicrobial peptide (AMP) that plays a key role in innate immunity of crustaceans. In this study, we cloned the entire 660 bp crustin-like sequence with a 507 bp open reading frame encoding a 168 amino acid from Marsupenaeus japonicus. The crustin-like gene was primarily expressed in gills and over-expressed in shrimp hemocytes after challenge with WSSV or Vibrio alginolyticus. After knockdown crustin-like gene using specific double-stranded RNA (CRU-dsRNA), IMD, Rab7, L-lectin, mitogen-activated protein kinase, p53, prophenoloxidase and Rho were down-regulated and nitric oxide synthase, myosin and tumor necrosis factor-α were up-regulated in hemocytes at 24 h post dsRNA treatment. After WSSV challenge, The mortality, WSSV copy number and expressions of WSSV immediate early genes (IE1, IE2, DNA polymerase, VP28) were both decreased but the apoptosis rate was increased in CRU-dsRNA-treated shrimps, indicating that WSSV may take advantage of crustin-like to benefit its replication. After silenced the crustin-like, the results of phagocytosis showed that the phagocytic rate of shrimp hemocytes on WSSV decreased significantly. In contrast, the absence of crustin-like in shrimps increased the mortality following V. alginolyticus challenge, indicating that crustin-like may play a positive role in the antibacterial process. The phagocytosis experiment showed there was a higher phagocytosis rate of hemocytes after CRU-dsRNA treatment. The result indicated that V. alginolyticus may be able to use crustin-like to avoid phagocytosis of shrimp hemocytes. These results further added to our understanding of the function of crustin-like peptide and also provided its potential role in innate immunity in shrimp.

In vitroassembly ofPenaeus monodondensovirus (PmDNV)-like particles produced in a prokaryote expression system

Viral diseases are a significant problem in the shrimp aquaculture industry as outbreaks can cause significant mortality and economic loss. While it has been shown that triggering the shrimp RNA interference pathway through dsRNA is a potentially viable treatment pathway, this approach is hampered by the lack of a suitable delivery mechanism. Virus-like particles (VLPs), which are structurally similar to native viruses but lack the genetic material, could possibly be developed as a delivery vehicle. To generate a candidate VLP, the Penaeus monodon densovirus (PmDNV) capsid protein was cloned with an added histidine tag and expressed in an E. coli expression system. While the protein was expressed in inclusion bodies, the recombinant PmDNV capsid protein could be dissolved and subsequently purified by nickel affinity column chromatography. The formation of VLP from this purified rPmDNV capsid protein was investigated by transmission electron microscopy, and PmDNV-VLPs were observed that looked similar to the native PmDNV virion. Our results suggest that the PmDNV-like particle could be promisingly applied towards vaccination and that this PmDNV-like particle can potentially serve as a system for delivery of nucleic acids to trigger innate immunity in shrimp.

Identification and functional analysis of a TEP gene from a crustacean reveals its transcriptional regulation mediated by NF-κB and JNK pathways and its broad protective roles against multiple pathogens

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.

Identification and functional characterization of a novel Spätzle gene in Litopenaeus vannamei

Shrimp innate immunity is the first line of resistance against pathogenic bacteria. The Toll-like receptor (TLR)-NF-κB pathway is vital in this immunity process. In this study, a novel Spätzle gene (LvSpz4) of Litopenaeus vannamei was cloned and functionally characterized. The open reading frame of LvSpz4 was 918 bp, which encoded a putative protein with 305 amino acids. LvSpz4 was most expressed in the gills of L. vannamei. This expression was induced by Vibrio alginolyticus or Staphylococcus aureus infection or lipopolysaccharide stimulation. The reporter gene assay showed that LvSpz4 could activate the promoters of Pen4, Drs, AttA, Mtk, and white spot syndrome virus immediate early gene1 in Drosophila Schneider 2 (S2) cells. Knockdown LvSpz4 increased the cumulative mortality of L. vannamei upon V. alginolyticus infection. The unfolded protein response (UPR) induced the expression of LvSpz4 in L. vannamei. Moreover, the promoter of LvSpz4 was activated by L. vannamei X-Box binding protein 1 and activating transcription factor 4 in S2 cells. These results suggested that LvSpz4 was involved in L. vannamei innate immunity and caused the crosstalk between the TLR-NF-κB pathway and UPR.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

Identification and functional characterizations of serpin8, a potential prophenoloxidase-activating protease inhibitor in Pacific white shrimp, Litopenaeus vannamei

Serpins have been characterized from varieties of organisms by their inhibitory roles on serine or cysteine proteases. However, research for the functional study of serpins in crustacean is relatively small. To fully clarify the immune characterizations of serpin, a novel serpin (named Lvserpin8) encoding 414 amino acids with a 19-amino acid signal peptide and a serpin domain was identified from the Pacific white shrimp Litopenaeus vannamei. Sequence analysis indicated that the genomic Lvserpin8 gene contains 5 exons and 4 introns. The P1 residues of the predicted scissile bond in the reactive center loop (RCL) region represented for Lysine (Lys), which is in accordance with Pmserpin8, Dmserpin27A, Ofserpin3, Bmserpin3 and Msserpin3. Quantitative results showed that high mRNA expression of Lvserpin8 was detected in hepatopancreas and testis. Notably, a significant increase of Lvserpin8 was appeared post injection of Vibrio anguillarum, and Micrococcus lysodeikticus. Moreover, Lvserpin8 was knocked down in vivo by double-stranded RNA (dsRNA) mediated RNA interference (RNAi). Suppression of Lvserpin8 led to a significant increase in the transcripts of LvPPAE2 (Prophenoloxidase-activating Enzyme 2) and cumulative mortality. What's more, recombinant Lvserpin8 protein (rLvserpin8) displayed inhibition roles on trypsin activity, and prophenoloxidase activation. Taken together, the results implied that Lvserpin8 may be involved in shrimp innate immunity via the inhibition of prophenoloxidase-activating proteases.

Involvement of a LysM and putative peptidoglycan-binding domain-containing protein in the antibacterial immune response of kuruma shrimp Marsupenaeus japonicus.

Lysin motif (LysM) is a peptidoglycan and chitin-binding motif with multiple functions in bacteria, plants, and animals. In this study, a novel LysM and putative peptidoglycan-binding domain-containing protein was cloned from kuruma shrimp (Marsupenaeus japonicus) and named as MjLPBP. The cDNA of MjLPBP contained 1010 nucleotides with an open reading frame of 834 nucleotides encoding a protein of 277 amino acid residues. The deduced protein contained a Lysin motif and a transmembrane region, with a calculated molecular mass of 31.54kDa and isoelectric point of 8.61. MjLPBP was ubiquitously distributed in different tissues of shrimp at the mRNA level. Time course expression assay showed that MjLPBP was upregulated in hemocytes of shrimp challenged with Vibrio anguillarum or Staphylococcus aureus. MjLPBP was also upregulated in hepatopancreas after white spot syndrome virus and bacteria challenge. The recombinant protein of MjLPBP could bind to some Gram-positive and Gram-negative bacteria and yeast. Further study found that rMjLPBP bound to bacterial cell wall components, including peptidoglycans, lipoteichoic acid, lipopolysaccharide, and chitin. The induction of several antimicrobial peptide genes and phagocytosis-related gene, such as anti-lipopolysaccharide factors and myosin, was depressed after knockdown of MjLPBP. MjLPBP could facilitate V.anguillarum clearance invivo. All the results indicated that MjLPBP might play an important role in the innate immunity of shrimp.

Molecular characterization and function of a PTEN gene from Litopenaeus vannamei after Vibrio alginolyticus challenge

PTEN, a tumor suppressor gene, suppresses cell survival, growth, apoptosis, cell migration and DNA damage repair by inhibiting the PI3K/AKT signaling pathway. In this study, the full-length Litopenaeus vannamei PTEN (LvPTEN) cDNA was obtained, containing a 5′UTR of 59bp, an ORF of 1269bp and a 3′UTR of 146bp besides the poly (A) tail. The PTEN gene encoded a protein of 422 amino acids with an estimated molecular mass of 48.3 KDa and a predicted isoelectric point (pI) of 7.6. Subcellular localization analysis revealed that LvPTEN was distributed in both cytoplasm and nucleus, and the tissue distribution patterns showed that LvPTEN was ubiquitously expressed in all the examined tissues. Vibrio alginolyticus challenge induced upregulation of LvPTEN expression. Moreover, RNAi knock-down of LvPTEN in vivo significantly increased the expression of LvAKT mRNA, while reducing that of the downstream apoptosis genes LvP53 and LvCaspase3. LvPTEN knock-down also caused a sharp increase in cumulative mortality, bacterial numbers, and DNA damage in the hemolymph of L. vannamei following V. alginolyticus challenge, together with a sharp decrease in the total hemocyte count (THC). These results suggested that LvPTEN may participate in apoptosis via the PI3K/AKT signaling pathway in L. vannamei, and play an important role in shrimp innate immunity.

Identification and pathogenicity of Vibrio parahaemolyticus isolates and immune responses of Penaeus (Litopenaeus) vannamei (Boone).

Five different Vibrio parahaemolyticus strains (SH8, SH108, SH58, AH5 and GD10) isolated from the hepatopancreas of moribund shrimp in farms of mainland China were identified and capable of inducing massive mortality of Penaeus (Litopenaeus) vannamei. The immersion challenge results with five isolates indicated variance of virulence, while only GD10 caused massive sloughing of tubule epithelial cells which was recognized as the most significant symptom of AHPND. Differences in immune responses were detected of P.vannamei during 48h post-infection (p.i.) by injection or immersion challenge with V.parahaemolyticus (SH8, SH108 and GD10) isolates. When injected SH8 and SH108 isolates, the expression of lysozyme (LSZ) showing statistically significant upregulation at 16 and 48h p.i. and that of Toll-like receptors (TLR) showed statistically significant upregulation at 48h p.i. When immersion challenge with the GD10 isolate, TLR were upregulated after 8h p.i. challenge with 10(4) cfumL(-1) ; however, LSZ was downregulated when challenged with 10(3) cfumL(-1) . The results suggested that LSZ and TLR serve as crucial molecular markers of innate immunity in shrimp against V.parahaemolyticus infection. LSZ is a vital marker for acute bacterial infection, while TLR serves as a crucial marker for chronic infection.

Identification and characterization of MKK7 as an upstream activator of JNK in Litopenaeus vannamei.

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 3093bp in length, with an open reading frame (ORF) of 1440bp 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.