Two types of calmodulin play different roles in Pacific white shrimp (Litopenaeus vannamei) defenses against Vibrio parahaemolyticus and WSSV infection.

Calmodulin (CaM) plays an important role in calcium-dependent signal transduction pathways. In the present study, two alternative splicing isoforms of CaM (named LvCaM-A and LvCaM-B) cDNA were cloned from the Pacific white shrimp, Litopenaeus vannamei. LvCaM-A was of 1101 bp, including a 5'-terminal untranslated region (UTR) of 70 bp, a 3'-terminal UTR of 581 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculated molecular weight (Mw) of 17 kDa and pI of 4.41. LvCaM-B was 689 bp, including a same 5'-UTR of 70 bp, a 3'-terminal UTR of 109 bp and an ORF of 510 bp encoding a polypeptide of 169 amino acids with a calculated Mw of 19 kDa and pI of 4.36. Both LvCaM-A and LvCaM-B contained 4 conservative EF-hand motifs. Quantitative real-time reverse transcription PCR analysis revealed LvCaM-A to be expressed in most shrimp tissues, with the predominant expression in nerve and the weakest expression in heart. However, LvCaM-B expression level was much weaker than those of LvCaM-A in all the tested tissues with main expression in hepatopancreas. The expression of LvCaM-A and LvCaM-B after challenge with Vibrio parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas and nerve. The results indicated that LvCaM-A and LvCaM-B transcripts could be significantly induced in hemocytes and hepatopancreas respectively by injection with V. parahaemolyticus. The highest expression of LvCaM-A was in the hemocytes with 2.3 times (at 48 h) greater expression than in the control (p < 0.05). However, sharp down-regulation of both LvCaM-A and LvCaM-B were detected in nerve after Vibrio- and WSSV injection (p < 0.05). These results suggested that CaM might play an important role in shrimp's defense against pathogenic infection.

Reactive oxygen system plays an important role in shrimp Litopenaeus vannamei defense against Vibrio parahaemolyticus and WSSV infection.

The present study investigated the in vivo hemocytic and hepatopancreatic response to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) injection in shrimp Litopenaeus vannamei. The proliferation of bacteria and virus in shrimp, animal mortality, total hemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst, and gene expression of immune factors associated with immune recognition (lectin), prophenoloxidase (proPO) activation, and the anti-microorganism (lysozyme) and active oxygen defense response (including respiratory burst, cytosolic manganese superoxide dismutase [C-MnSOD], and catalase [CAT]) were quantified. Shrimp death rate increased significantly and was time-dependent after V. parahaemolyticus or WSSV injection. The production of superoxide anion, and the gene expression including lectin in hemocytes, proPO in the hepatopancreas, lysozyme, C-MnSOD and CAT could be induced by injection with V parahaemolyticus and WSSV. The highest value of lysozyme was in the hemocytes with 66.59 times (at 3 h) greater expression than in the control group after WSSV injection and 3.69 times (24 h) greater than in the control group after V parahaemolyticus injection. In the hepatopancreas, CAT expression showed a significant increase, with up to 16 times greater expression than in the control group at 6 h postinjection with WSSV and 7.02 times greater expression than in the control group at 48 h postinjection with V parahaemolyticus (p < 0.05). However, significant decreases in PO activity and proPO transcripts in hemocytes and lectin transcripts in the hepatopancreas were detected after V parahaemolyticus and WSSV injection (p < 0.05). The results suggest that lysozyme, the antioxidase system, and reactive oxygen species might play a crucial role in shrimp defense against bacterial and viral infection.

Molecular cloning and expression of NOS in shrimp, Litopenaeus vannamei.

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in recent years. However, the lack of sequence information and clones of shrimp NOS cDNA limits further study on its characterization and function in this species. In this report, the cDNA of NOS contained full-length ORF was cloned from the Pacific white shrimp, Litopenaeus vannamei. It was of 4680 bp, including a 5'-terminal untranslated region (UTR) of 278 bp, a 3'-terminal UTR of 862 bp, which contained 5 ATTTA repeats, and an open reading frame (ORF) of 3540 bp encoding a polypeptide of 1179 amino acids. It contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time reverse transcription PCR analysis revealed L. vannamei NOS (LvNOS) to be expressed in most shrimp tissues, with highest expression in the hepatopancreas and weakest expression in skin. The expression of LvNOS after challenge with LPS and poly I:C was tested in hemocytes, hepatopancreas and nerve. The results indicated that the NOS transcript level could be induced in hemocytes by injection with LPS. The highest expression was in the hemocyte, with 8.8 times (at 3 h) as much as that in the control (p < 0.05). However, sharp down-regulation of NOS was found in hepatopancreas and nerve after LPS and poly I:C injection (p < 0.05). These results suggested that NOS might play an important role in shrimp's defense against pathogenic infection.

Immune response and gene expression in shrimp (Litopenaeus vannamei) hemocytes and hepatopancreas against some pathogen-associated molecular patterns.

The effects of some pathogen-associated molecular patterns (PAMPs) (laminarin, LPS and poly I:C) on total hemocyte counts (THC), phenoloxidase (PO) activity, superoxide anion production and lectin, prophenoloxidase, lysozyme, cytosolic manganese superoxide dismutase (C-MnSOD) and catalase (CAT) gene expression were studied. The results showed that the production or activity of most tested immune factors and the expression of most tested genes were up-regulated after stimulation with PAMPs, among which the highest value of lectin with 4.4 times as much as that of the control group appeared at 6 h in hemocytes, of CAT with 47 times as much as that of the control group appeared at 12 h in hepatopancreas, and with 2.7 times higher than that of the control group at 24 h of C-MnSOD in hepatopancreas after laminarin injection. The peak value of proPO, lysozyme and C-MnSOD appeared at 6 h in hepatopancreas, 24 h in hepatopancreas and 24 h in hemocytes after LPS injection, respectively. The highest expression level of lysozyme appeared at 12 h in hemocytes after poly I:C injection. However, significant decreases of PO activity in hemocytes and lectin expression in hepatopancreas were found after poly I:C injection, and a dramatic down-regulation of proPO expression from 3 h to 48 h was found in hemocytes after injection with laminarin, LPS and poly I:C. The results suggest that the shrimp immune response could be activated or inhibited by different PAMPs, and that the hepatopancreas also plays a key role by synthesizing immune factors.

Arginine kinase from Litopenaeus vannamei: cloning, expression and catalytic properties.

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the full-length cDNA of AK was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Escherichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps.

Molecular cloning and expression of MyD88 in large yellow croaker, Pseudosciaena crocea.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). In this report, the full-length cDNA of MyD88 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1574 bp, including a 5'-terminal untranslated region (UTR) of 89 bp, a 3'-terminal UTR of 621bp and an open reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids. It contained a typical death domain at the N-terminal and a conservative Toll/IL-1R (TIR) domain structure at the C-terminal. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of MyD88 with the highest expression in the spleen and the weakest expression in the muscle. The expression of MyD88 after challenge with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. It suggested that the highest expression was in the spleen (p<0.05) with 1.9 times (at 48 h) as much as that in the control and the lowest expression of MyD88 was in the liver (p<0.05) with 0.29 times (at 3h) of that in the control. These results indicated that as a universal key adaptor in the Toll-like receptor pathway in mammals, MyD88 might play an important role in large yellow croaker defense against pathogenic infection.

Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker, Pseudosciaena crocea.

Toll-like receptors (TLRs) are the archetypal pattern-recognition receptors in sensing foreign pathogens. In this report, two alternative splicing isoforms of TLR9 (named PcTLR9A and PcTLR9B) cDNA were cloned from the large yellow croaker, Pseudosciaena crocea. The full-length cDNA of PcTLR9A was of 3637bp, including a 5'-terminal untranslated region (UTR) of 111bp, 3'-terminal UTR of 355bp and an open reading frame (ORF) of 3171bp encoding a polypeptide of 1056 amino acids. However, the full-length cDNA of PcTLR9B was 119bp longer than that of PcTLR9A from the position of 3079-3197bp, which encoded a polypeptide of 1006 amino acids. Both of the PcTLR9A and PcTLR9B contained 12 typical structures of leucine-rich repeats (LRRs), an LRRTYP and an LRRCT in the extracellular region and a conservative Toll/IL-1R (TIR) domain in the intracellular region. However, compared to PcTLR9A, conservative Box 3 was absent in PcTLR9B TIR domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR9A and PcTLR9B with the highest expression in spleen and the weakest expression in muscle. Expression of PcTLR9A and PcTLR9B after stimulation with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. This indicated that the highest expression was 3.3 times (at 24h) as much as that in the control in the spleen (p<0.05) and the lowest expression of PcTLR9A was 1/4 times (at 3h) of that in the control in the liver (p<0.05). PcTLR9B showed a similar expression pattern to PcTLR9A post-injection. These results suggested that both PcTLR9A and PcTLR9B might play an important role in large yellow croaker defense against pathogenic infection.