Identification and functional study of a shrimp Dorsal homologue.

Rel/NF-kappaB transcription factors play central roles in induction and regulation of innate immune responses. Here, identification and functional analysis of LvDorsal, a Dorsal homologue from the Pacific white shrimp Litopenaeus vannamei, were described. The full-length cDNA of LvDorsal is 2204bp with an open reading frame that encodes 400 amino acids. The deduced LvDorsal contains a conserved Rel homology domain (RHD), an IPT (Ig-like, plexins and transcription factors) domain and a nucleus localization signal, suggesting that it belongs to the class II NF-kappaB. RT-PCR analysis showed that LvDorsal mRNAs were expressed in all the tissues tested, including gill, epidermis, hemocytes, intestine, stomach, eyestalk, brain, hepatopancreas, muscle, heart and pyloric caecum. Immunofluorescence assay showed that recombinant LvDorsal was translocated into the nucleus of Drosophila S2 cells. Electrophoretic mobility shift assay illustrated that recombinant LvDorsal RHD from S2 cells bound specifically with D. melanogaster kappaB motifs. Additionally, the dual-luciferase reporter assays indicated that LvDorsal could transactivate the reporter gene controlled by the 5' flanking region of shrimp penaeidin-4 and Drosophila attacin genes, suggesting that LvDorsal can regulate the transcription of shrimp penaeidin-4 gene. Study of LvDorsal will help us to better understand shrimp immunity and may help to obtain more effective methods to prevent shrimp diseases.

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.

A novel C-type lectin from the shrimp Litopenaeus vannamei possesses anti-white spot syndrome virus activity.

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 (Glu(99)-Pro(100)-Asn(101)) 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.

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).

Characterization of a prophenoloxidase from hemocytes of the shrimp Litopenaeus vannamei that is down-regulated by white spot syndrome virus.

Previously, a prophenoloxidase (proPO) gene (named proPO-a here) from hemocytes of Litopenaeus vannamei was isolated. Here, a proPO-b gene was also identified and characterized from hemocytes of L. vannamei. The cDNA sequences of proPO-a and proPO-b were compared, and it was found that both proPOs had a microsatellite DNA site near the 3' end of the open reading frame (ORF). However, the microsatellite DNA of proPO-b contained a compound imperfect simple sequence repeats (SSR) ((CT)(38)(CA)(8)(AA)(CA)(3)(TA)(CA)(14)), which was different from the perfect one ((CT)(20)) of proPO-a, and the cDNA sequences of proPO-a and proPO-b prior to the microsatellite DNA were almost identical, but differed after the microsatellite DNA. ProPO-b (3232 bp) was longer than proPO-a (2471 bp). The 3' UTR sequence after SSR of proPO-a was not detected in shrimp randomly collected from five different geographically separate populations by reverse-transcription polymerase chain reaction (RT-PCR). On the contrary, the 3' UTR sequence of proPO-b was detected in all five groups of shrimps. Northern blot analysis showed that a transcript at approximately 3.2kb, but not 2.5kb, was detected mainly in hemocytes, and also present in midgut, gill, heart, stomach, posterior midgut cecum, and cuticular epidermis, but no signal was detected in hepatopancreas and musculature. RT-PCR and quantitative real-time RT-PCR analysis showed similar results of the proPO-b expression profile in these shrimp tissues. We also observed that proPO-b expression was down-regulated in shrimp challenged with white spot syndrome virus (WSSV). Our results suggest that proPO-b is a main transcript form of proPO gene in L. vannamei, and it may play a role in defence against WSSV virus.

Profiling of differentially expressed genes in hepatopancreas of white spot syndrome virus-resistant shrimp (Litopenaeus vannamei) by suppression subtractive hybridisation.

In order to find immune-relevant factors responsible for virus resistance and response to the virus infection, the suppression subtractive hybridisation method was employed to identify differentially expressed genes and their expression profiles in the hepatopancreas of the white spot syndrome virus (WSSV) resistant and susceptible Pacific white shrimp (Litopenaeus vannamei). Two forward subtractive libraries (at 0 and 48h time point) and two reverse subtractive libraries (at 0 and 48h time point) were constructed, and more than 1200 clones were sequenced, of which 40 differentially expressed genes were identified. These genes encode proteins corresponding to a wide range of functions, including defence-related proteins, enzymes, transcription factors, apoptotic-related proteins, intracellular components potentially related to signaling cascades, metabolic proteins, and cytoskeletal protein. Five genes (laccase, carboxypeptidase B, H(+)-transporting ATP synthase, Acyl-ConA-binding protein (ACBP), and cortical granule protein with LDL-receptor) are found for the first time in shrimp and their expressions were up-regulated in the virus-resistant shrimp. Among the 40 genes, 30 showed up-regulation in the virus-resistant shrimp comparing with susceptible shrimp, while 10 genes showed down-regulation. Haemocyanin was the most abundant gene in our forward subtractive libraries. In addition, chathepsin L, ecdysteroid regulated protein, zinc proteinase, lectin, sterol carrier protein-X, lysozyme, cortical granule protein with LDL-receptor, leucine-rich repeat LGI family, fatty acid binding protein, and preamylase all showed up-regulation in the resistant shrimp. Furthermore, a number of genes encoding apoptotic-related proteins and antioxidant enzymes were expressed at a higher level in the virus-resistant shrimp. The high expression of the immune-relevant genes in response to the virus infection provides a new insight for further study in the shrimp innate immunity.