Chinese White Shrimp Fenneropenaeus Chinensis Research Articles

A study on β-glucan binding protein (β-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus

The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiβ-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiβ-GBP had the specific binding site for soluble or insoluble β-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiβ-GBP activating PO were 40°C and pH 7–8. Moreover, even at 100°C Fiβ-GBP enhanced PO activity highlighting that Fiβ-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiβ-GBP significantly promoted the PO activity in presence of Mg2+ and Ca2+. The breakdown of para nitroanilide from Nα−Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiβ-GBP and also increased concentration of Fiβ-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.

Study on the genetic variability of the hatchery-released and wild populations of Chinese white shrimp Fenneropenaeus chinensis in the Yellow Sea and Bohai Sea

In relation to the stock enhancement program for Chinese white shrimp Fenneropenaeus chinensis, hatchery-released juveniles and wild samples in the Yellow Sea and Bohai Sea were genotyped using amplified fragment length polymorphism (AFLP) markers to investigate the levels of genetic variation among these populations. Five AFLP primer combinations generated a total of 757 bands across the 268 individuals in nine hatchery-released populations and three wild populations. All 757 bands were polymorphic. High polymorphism at AFLP markers was found within both hatchery-released and wild populations. The percentage of polymorphic loci for populations ranged from 87.22 to 98.60%, while the Nei’s genetic diversity ranged from 0.1822 to 0.2728. Compared with wild populations, the nine hatchery-released populations showed significant genetic changes based on F ST values. All populations were divided into two distinct groups by principal coordinate analysis (PcoA) analysis. Three wild populations (Dalian, Kengli, Middle Yellow Sea) with hatchery-released populations from Rongcheng, Panjin, and Dandong clustered as one group; the rest of hatchery populations formed another group. Interestingly, no obvious geographic cluster was found in the unweighted pair group method with arithmetic mean tree and PcoA. We recommend monitoring the genetic variability of selected hatchery populations to enhance the conservation of natural Chinese white shrimp F. chinensis resources.

Ecological effects of co-culturing the sea cucumber Apostichopus japonicus with the Chinese white shrimp Fenneropenaeus chinensis in an earthen pond

Using net enclosures in an earthen pond, we established three culture treatments with the sea cucumber Apostichopus japonicus and the Chinese white shrimp Fenneropenaeus chinensis: monoculture of sea cucumbers (C), monoculture of shrimp (S), and co-culture of the two species (CS). We measured levels of suspended particulate matter in the water column; total organic matter, total organic carbon, total nitrogen, and carbon/nitrogen ratios in both settling particles and the sediment; and chlorophyll a levels in the sediment. We then compared these variables between the three treatments. We also examined growth, survival, and yield of the two species in the different treatments. From June to September, the mean monthly suspended particulate matter sedimentation rates in the CS and S treatments were significantly (P<0.05) greater than those in the C treatment. From August to November, the mean monthly total organic matter, total organic carbon, total nitrogen, and chlorophyll a contents in the sediment in the CS and S treatments were significantly ( P <0.05) greater than those in the C treatment. Final wet weight, specific growth rate, survival rate, and total yield of sea cucumbers in co-culture were all significantly greater than those of sea cucumbers in monoculture. There were no significant differences among any of these variables for shrimp reared in the two systems. The bioturbation of the sediment and fecal production of the shrimp likely supplied natural food for the sea cucumbers. Co-culture of the two species is a viable option for increasing yield per unit area, maximizing use of the water body, and diversifying crop production.

C-type Lectin Binds to β-Integrin to Promote Hemocytic Phagocytosis in an Invertebrate

Phagocytosis is a conserved cellular response among metazoans. Opsonins are some molecules that label targets to increase their susceptibility to phagocytosis. Opsonins are usually captured by receptors on the surface of phagocytes. Our previous study found the C-type lectin FcLec4 from Chinese white shrimp Fenneropenaeus chinensis might function as an opsonin to facilitate bacterial clearance. In the present study we purified the native FcLec4 protein and confirmed its opsonic activity in the near relation, kuruma shrimp Marsupenaeus japonicus. The possible receptor of FcLec4 was identified as β-integrin by panning a T7 phage display library of shrimp hemocytes and then confirmed by co-immunoprecipitation assay. We further proved that the interaction between FcLec4 and β-integrin did not rely on the carbohydrate recognition domain but on the N terminus of FcLec4. In addition, inhibition of FcLec4 expression using RNAi delayed bacterial clearance, and β-integrin knockdown suppressed the opsonic activity of FcLec4. This study is the first to show the direct interaction between an opsonin and its receptor in crustaceans. Our study provides new insights into invertebrate phagocytosis and the functions of C-type lectins.

Characterization of an immune deficiency homolog (IMD) in shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkii)

The immune deficiency (IMD) signal pathway mediates immunity against Gram-negative bacteria in Drosophila. Recent studies show that the IMD pathway also involves in antiviral innate immune responses. The functions of the pathway in crustacean immunity are largely unknown. In this paper, two IMDs (FcIMD and PcIMD), one of the key elements of the IMD pathway, were identified from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Both proteins have a death domain located at the C-terminal. FcIMD was mainly expressed in the gills and stomach and PcIMD was mainly detected in the heart, hepatopancreas, and stomach. FcIMD peaked in hemocytes at 12h after white spot syndrome virus (WSSV) challenge and it peaked in the gills at 6h after WSSV challenge, but it was decreased at 2h and kept the low level to 24h in hemocytes and no obviously change in gill after Vibrio anguillarum challenge. PcIMD first decreased in hemocytes at 2h and peaked at 12h in hemocytes after V. anguillarum challenge. It was also upregulated in gill after bacterial challenge, peaked at 2h, and decreased at 6h, and then gradually increased at 12–24h. PcIMD has no significant change in hemocytes and gill after WSSV challenge. Western blot analysis detected FcIMD protein in all tissues, and immunocytochemical analysis localized FcIMD in the cytoplasm of hemocytes. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of three kinds AMP genes, including crustins, anti-lipopolysaccharide factors and lysozymes, in shrimp and crayfish. They are Cru 1, Cru 2, ALF 1, ALF 2 and Lys 1 in crayfish, and Cru1, Cru 3, ALF 6, ALF 8, and Lys2 in shrimp. These results suggest that although IMD distribution and expression patterns have some differences, the IMD pathway may have conserved function for AMP regulation in shrimp and crayfish immunity against Gram-negative bacteria.

A novel pathogen-binding gC1qR homolog, FcgC1qR, in the Chinese white shrimp, Fenneropenaeus chinensis

In vertebrates, the globular “head” of complement component C1q receptor (gC1qR) is a versatile, multiligand binding protein. However, research on its function in invertebrates is limited. In the present study, a full-length cDNA sequence of a novel gC1qR homolog, FcgC1qR, from the Chinese white shrimp Fenneropenaeus chinensis was cloned. Semi-quantitative polymerase chain reaction (PCR) detected FcgC1qR in all examined tissues, with the highest level detected in the intestine. Western blot assay further revealed that the FcgC1qR protein was distributed in all tested tissues except the cell-free hemolymph of normal Chinese white shrimp. In the expression pattern study, quantitative real-time PCR demonstrated that the transcripts of FcgC1qR were up-regulated when challenged with bacteria ( Vibrio anguillarum or Staphylococcus aureus) and white spot syndrome virus. Subsequently, FcgC1qR was over-expressed in Escherichia coli, and the polyclonal antibody was prepared with the purified recombinant protein. Microorganism binding was examined using Western blot assay, and revealed that FcgC1qR could bind to Bacillus cereus, Bacillus thuringiensis, S. aureus, V. anguillarum, Vibrio harveyi, and Candida albicans. FcgC1qR was also proven able to bind to S. aureus in a concentration-dependent manner, and this binding activity was partly inhibited by the polyclonal antibody. These results suggest that FcgC1qR may be involved in defending against bacterial infections in the Chinese white shrimp.

Cloning and characterization of Rap GTPase from the Chinese white shrimp Fenneropenaeus chinensis

Ras-related protein Rap GTPase has been implicated in cell adhesion, cell proliferation, and cell junction formation. The first shrimp Rap cDNA ( FcRap) was recently identified from the Chinese white shrimp Fenneropenaeus chinensis. The full length of FcRap is 1013 bp, with a 561 bp open reading frame that encodes a 186 amino acid protein. FcRap has a calculated molecular mass of 20.90 kDa and p I of 6.37. Phylogenetic analysis shows that FcRap and other Rap proteins are clustered into one group. Results from the quantitative real-time polymerase chain reaction show that FcRap could be detected mainly in the hemocytes, hepatopancreas, stomach, and gills, whereas a relatively lower expression level could be detected in the heart and intestines. FcRap in the hemocytes was upregulated 2 h post Vibrio challenge, and it was upregulated 2 h post white spot syndrome virus (WSSV) challenge, and peaked at 6 h before it declined at 12 h. No variation in the FcRap transcript was observed in the gills under the Vibrio challenge, but it was initially downregulated 2 h post WSSV challenge, and then it was upregulated and peaked at 6 h before it was eventually went down at 12 h. The rFcRap protein was successfully expressed in Escherichia coli BL21DE3. The pull-down analysis showed that rFcRap protein could interact with VP28, an envelope protein of WSSV. The probable roles of Rap GTPase in shrimp innate immunity are presented for the first time.

Molecular cloning and expression analysis of signal transducer and activator of transcription (STAT) from the Chinese white shrimp Fenneropenaeus chinensis

Innate immunity is the first line of defense by a host against invading pathogens. Several signaling pathways participate in the immune response, one of which is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Various evidences have been provided to suggest that the JAK/STAT pathway is involved in both antibacterial and antiviral immunities. In this study, the full-length cDNA and gene sequence of STAT (designated as FcSTAT) was cloned from the Chinese white shrimp, Fenneropenaeus chinensis. Phylogenetic analysis reveals that the FcSTAT is clustered with STAT5s and STAT6s from vertebrates and STATs from invertebrates. Quantitative real-time PCR exhibited that the FcSTAT had a wide distribution in all detected tissues and developmental stages. Time course analysis of the transcription level after WSSV challenge showed a noticeably early up-regulation of FcSTAT in hemocytes, hepatopancreas, and intestines. The expression levels of FcSTAT increased corresponding to Vibrio anguillarum stimulation in both hemocytes and hepatopancreas as well. All these imply that the JAK/STAT pathway participates in the immune response against bacteria and virus in F. chinensis.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to white spot syndrome virus

White spot syndrome virus (WSSV) infects all shrimp species and is the greatest detriment to shrimp culture. To better understand the mechanism of molecular responses to WSSV infection in Chinese white shrimp Fenneropenaeus chinensis, two dimensional electrophoresis (2-DE) was used. Differentially expressed proteins in the hepatopancreas of control and WSSV-injected Chinese white shrimp between (6, 12 and 24 h post-injection) were screened. Quantitative intensity analysis and mass spectrometry revealed that 54 spots of 47 proteins were significantly up-regulated, including some immune-related proteins, such as Toll receptor precursor, Leu-rich repeat protein, peroxinectin and serine proteinase-like protein. Fourteen spots of 13 proteins, such as heat shock protein, ATP synthase sub-unit beta and thrombospondin, were down-regulated in WSSV-infected shrimps. Protein expression patterns of the infected shrimp were drastically altered by WSSV infection. Some of the altered proteins were further investigated at the mRNA level using semi-quantitative reverse transcript PCR. These data may provide some information about shrimp proteins that participate in the WSSV infection process.

Two cysteine proteinases respond to bacterial and WSSV challenge in Chinese white shrimp Fenneropenaeus chinensis

The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis ( FcCathL, FcLegu) were obtained. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. Time-course analysis of FcCathL showed that FcCathL was upregulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12 h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2 h to 6 h and then recovered from 12 h to 24 h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time.

A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin (FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca2+-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca2+-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp.

Molecular cloning and characterization of three crustins from the Chinese white shrimp, Fenneropenaeus chinensis

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences ( Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.

A C-type lectin is involved in the innate immune response of Chinese white shrimp

C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.

A novel C-type lectin (FcLec4) facilitates the clearance of Vibrio anguillarum in vivo in Chinese white shrimp

C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps.