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.

Ji PF ，Yao CL ，Wang ZY 《-》

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.

Yao CL ，Ji PF ，Wang ZY ，Li FH ，Xiang JH ... -  《-》

Molecular and immune response characterizations of a novel AIF and cytochrome c in Litopenaeus vannamei defending against WSSV infection.

Apoptosis inducing factor (AIF) and cytochrome c (CYC) are two mitochondrial apoptogenic factors. In the present study, the cDNA sequences of AIF (LvAIF) and CYC (LvCYC) were cloned from Pacific white shrimp, Litopenaeus vannamei. The LvAIF was 1664 bp, including a 5'-terminal untranslated region (UTR) of 154 bp, an open reading frame (ORF) of 1323 bp encoding a polypeptide of 440 amino acids (aa) and a 3' UTR of 187 bp. The LvCYC was 582 bp, including a 50 bp 5' UTR, a 315 bp ORF encoding for 104 aa, and a 217 bp 3' UTR. The deduced protein of LvAIF contained a conserved Pyr_redox and AIF_C domain at the N-terminal and the predicted LvCYC included a conservative cytochrome_C domain, respectively. Phylogenetic analysis revealed that LvAIF belonged to AIF1 subfamily and showed a close relationship with AIF1 from vertebrates and LvCYC showed the closest relationship with its counterparts from shrimp Marsupenaeus japonicus. Tissue expression profiles showed that both LvAIF and LvCYC existed in most tissues, with the most predominant expression of LvAIF in intestine, then followed muscle and the weakest expression in gill. The highest expression of LvCYC was detected in muscle, and the weakest expression was in hemocytes. Additionally, after white spot syndrome virus (WSSV) infection, the significant up-regulation of LvAIF, LvCYC and caspase 3 transcripts and the increase of pro-caspase 3 and active-caspase 3 protein were detected at most time points (P < 0.05). However, all of the three genes down-regulated in hemocytes in the early stage after WSSV infection. WSSV proliferation and shrimp mortality showed a time-dependent manner and the production of ROS in hemocytes were significantly increased at 6 and 24 h after infection. Our results showed that the apoptotic genes AIF, CYC and caspase 3 might play crucial roles in hepatopancreas, however, the production of ROS in hemocytes might be important in shrimp defense against WSSV infection.

Hu WY ，Yao CL 《-》

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

Ji PF ，Yao CL ，Wang ZY 《DISEASES OF AQUATIC ORGANISMS》

cDNA cloning, identification, tissue localisation, and transcription profile of a transglutaminase from white shrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus.

Yeh MS ，Liu CH ，Hung CW ，Cheng W ... -  《-》