Zhi-Xin Yin

A Novel C-Type Lectin from the Shrimp Litopenaeus vannamei Possesses Anti-White Spot Syndrome Virus Activity

ABSTRACT C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu99-Pro100-Asn101) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.

An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes

Invertebrates rely on innate immunity as the first line defense against microbes. In Drosophila, the inducible antimicrobial peptides (AMPs) regulated by the Toll and immune deficiency (Imd) pathways are important effectors in innate immunity. Here we report an immune deficiency homolog (LvIMD) from the white shrimp, Litopenaeus vannamei. The full-length cDNA of LvIMD is 758 bp with an open reading frame of 483 bp that encodes a putative protein of 160 amino acids including a death domain at the C-terminus. LvIMD death domain shows similarity to that of Drosophila IMD and human receptor interacting protein 1 (RIP1) of the tumor necrosis factor receptor (TNFR) pathway, with 27.9% and 26.4% identity, respectively. Phylogenetic analysis shows that LvIMD clusters with a predicted protein from the starlet sea anemone (Nematostella vectensis) independent to insect IMDs and vertebrates RIP1s. LvIMD mRNA is expressed in most tissues and is induced in hepatopancreas and hemocytes after immune challenge. Luciferase reporter assays confirm that LvIMD is able to induce the expression of AMP genes, including Drosophila Attacin A and shrimp Penaeidin 4 in S2 cells. To our knowledge, this is the first report that LvIMD participates in innate signaling to activate the expression of AMP genes in shrimp.

A novel prophenoloxidase 2 exists in shrimp hemocytes

The prophenoloxidase (proPO)-activating system in crustaceans and other arthropods is regarded as a constituent of the immune system and plays an important role in defense against pathogens. Hitherto in crustaceans, only one proPO gene per species has been identified. Here we report the identification of a novel proPO-2 (LvproPO-2) from the hemocytes of Litopenaeus vannamei, which shows 72% identity to proPO-1 (LvproPO-1) cloned previously. Northern blotting analysis and quantitative real-time PCR reveal that LvproPO-2 is mainly expressed in the hemocytes, and its expression is down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Western blotting analysis shows that most LvproPO-2/LvPO-2 (L. vannamei phenoloxidase-2) exists in the hemocytes, but not in plasma of L. vannamei. LvproPO-2/LvPO-2 could be detected on the hemocyte surface and the nucleus of hemocytes by indirect immunofluorescence assay (IFA). These findings provide insight into the molecular biological basis for further studying on the defense mechanism of shrimp innate immunity, especially on the proPO-activating system and melanization cascade of shrimp.

Cloning of IRAK1 and its upregulation in symptomatic mandarin fish infected with ISKNV.

Abstract Interleukin-1 receptor activated kinases (IRAKs) play crucial roles in the Toll-like receptor (TLR) mediated signal transduction pathways that control host innate immune responses. Here we report the cloning of an IRAK1 cDNA (named ScIRAK1) from the mandarin fish. The predicted ScIRAK1 peptide contains a death domain and a serine/threonine-specific kinase domain. Quantitative RT-PCR showed that ScIRAK1 mRNA was primarily expressed in blood cells and posterior kidney. Seven days following infection with infectious spleen and kidney necrosis virus (ISKNV), the ScIRAK1 mRNA level was significantly higher in the blood cells of clinically symptomatic fish than in the blood cells of asymptomatic fish or control fish injected with phosphate-buffered saline. Additional experiments showed that overexpression of ScIRAK1 in the 293T cells could induce NF-κB activation. These results suggest that ScIRAK1 may play a role in the pathology of ISKNV infection in the mandarin fish.

Changes in mortality and immunological variables of Litopenaeus vannamei parents and their filial families infected with white spot syndrome under different experimental conditions.

In order to find changes in mortality and immunological variables of Litopenaeus vannamei parents and the filial WSSV-resistant and -susceptible families after infection with WSSV under different experimental conditions, the haemolymph total haemocyte count (THC), phenoloxidase (PO), and superoxide dismutase (SOD) activities were measured at days 0, 1, 3, 6, 9, 12 and 15 after challenge and shrimp mortality was also recorded. When shrimps were challenged with 10(-3) (1.29x10(6)copiesmL(-1)), 10(-4) (1.29x10(5)copiesmL(-1)) or 10(-5) (1.29x10(4)copiesmL(-1)) WSSV stock solution (0.1mLshrimp(-1)), the cumulative mortalities (mean+/-S.E.) on day 15 were 100+/-0%, 79.3+/-1.1%, and 21.7+/-2.3%, respectively. Among shrimps challenged with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)), the cumulative mortalities (mean+/-S.E.) on day 15 in high-density (100shrimpsm(-3)), middle-density (50shrimpsm(-3)), and low-density (25shrimpm(-3)) groups were 95.5+/-0%, 84.7+/-0%, and 72.3+/-0%, respectively. The immunological variables including THC, PO, and SOD were decreased significantly at the beginning of infection stage, while these immunological variables for survivors reached almost the similar levels to the non-infection control group on day 15 after challenge with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)). Cumulative mortality (mean+/-S.E.) on day 15 in 17 filial families (G(2)) ranged from 13.3+/-1.9% to 100+/-0% when shrimps were challenged with 10(-4) (1.29x10(5)copiesmL(-1)) WSSV dilution (0.1mLshrimp(-1)). Although, the PO and SOD activities for shrimps in the WSSV-resistant family were slightly higher than those in the WSSV-susceptible family at the same sampling time after infection, these differences were not significant (p<0.05).