Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5'-terminal untranslated region (UTR) of 60 bp and a 3'-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys(53), Cys(128), Cys(144), Cys(152)) involved in the formation of disulfides bridges and the potential Ca2+/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca2+, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei. (C) 2008 Elsevier Ltd. All rights reserved.

Analysis of tandem repeats in the genome of Chinese shrimp Fenneropenaeus chinensis

Through random sequencing, we found a total of 884000 base-pairs (bp) of random genomic sequences in the genome of Chinese shrimp (Fenneropenaeus chinensis). Using bio-soft Tandem Repeat Finder (TRF) software, 2159 tandem repeats were found, in which there were 1714 microsatellites and 445 minisatellites, accounting for 79.4% and 20.6% of repeat sequences, respectively. The cumulative length of repeat sequences was found to be 116685 bp, accounting for 13.2% of the total DNA sequence; the cumulative length of microsatellites occupied 9.78% of the total DNA sequence, and that of minisatellites occupied 3.42%. In decreasing order, the 20 most abundant repeat sequence classes were as follows: AT (557), AC (471), AG (274), AAT (92), A (56), AAG (28), ATC (27), ATAG (27), AGG (18), ACT (15), C (11), AAC (11), ACAT (11), CAGA (10), AGAA (9), AGGG (7), CAAA (7), CGCA (6), ATAA (6), AGAGAA (6). Dinucleotide repeats, not only in the aspect of the number, but also in cumulative length, were the preponderant repeat type. There were few classes and low copy numbers of repeat units of the pentanucleotide repeat type, which included only three classes: AGAGA, GAGGC and AAAGA. The classes and copy numbers of heptanucleotide, eleven-nucleotide and thirteen-nucleotide primer-number-composed repeats were distinctly less than that of repeat types beside them.

Four viral proteins of white spot syndrome virus (WSSV) that attach to shrimp cell membranes

White spot syndrome virus (WSSV) is a major shrimp pathogen that has a widespread negative affect on shrimp production in Asia and the Americas. It is known that WSSV infects shrimp cells through viral attachment proteins (VAP) that bind with shrimp cell receptors. However, the identity of both WSSV VAP and shrimp cell receptors remains unclear. We used digoxigenin (DIG)labeled shrimp hemocyte and gill cell membranes to bind to WSSV proteins immobilized on nitrocellulose membranes, and 4 putative WSSV VAP (37 kDa, 39 kDa and 2 above 97 kDa) were identified. Mass spectrometric analysis identified the 37 kDa putative VAP as the product of WSSV gene VP281.

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a similar to6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use. (C) 2004 Elsevier Inc. All rights reserved.

Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp (Fenneropenaeus chinensis)

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3' and 5' complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleus tridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

Molecular cloning and response to laminarin stimulation of arginine kinase in haemolymph in Chinese shrimp, Fenneropenaeus chinensis

Arginine kinase (AK) was previously reported as a phosphagen-ATP phosphotransferase found in invertebrates. In this study, an 1184 bp cDNA was cloned and sequenced. It contained an open reading frame of 1068 bp that coded for 356 deduced amino acids of AK in Fenneropenaeus chinensis. The calculated molecular mass of AK is 40129.73 Da and pI is 5.92. The predicted protein showed a high level of identity to known AK in invertebrates and creatine kinase from vertebrates, which belong to a conserved family of ATP:guanidino phospho-transferases. In addition, AK protein in plasma of F. chinensis was identified using two-dimensional electrophoresis (2DE) and electrospray ionization mass spectrometry (ESI-MS) according to the calculated molecular mass and pI. AK was significantly decreased in the plasma of F. chinensis at 45 min and recovered at 3 It after laminarin injection as confirmed by 2DE and ESI-MS. The results showed that AK was one of the most significantly changed proteins on two-dimensional gel in the plasma proteins of F. chinensis at 45 min and 3 It after simulation. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

Purification and partial characterization of Mn superoxide dismutase from muscle tissue of the shrimp Macrobrachium nipponense

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

Primary culture and characteristic morphologies of medulla terminalis neurons in the eyestalks of Chinese shrimp, Fenneropenaeus chinensis

A method for culturing medulla terminalis (MT) neurons in the eyestalk of Chinese shrimp, Fenneropenaeus chinensis, was first established. The neurons showed immediate outgrowth in the culture medium supplemented with glutamine, glucose and antibiotics. The cells grew for about 2-7 days and then sustained for a week or more. At least six types of neurons were distinguished on the basis of size and form of soma and outgrowth pattern of cells. (C) 2003 Elsevier Science B.V. All rights reserved.

Gonad development characteristics and sex ratio in triploid chinese shrimp (Fenneropenaeus chinensis)

This paper details for the first time the gonad development characteristics and sex ratio of triploid shrimp (Fenneropenaeus chinensis). In triploid shrimp the development of gonad is apparently impaired, especially in females. In the ovary of triploids, germ cells mainly remain at oogonia stage during September through December. From January to February of the next year, partial primary oocytes developed in the ovary lobes. Spermatocytes and spermatids could be observed in the testes of triploids, and a few sperm were observed in the vas deferens and spermatophores. The morphology of sperms in triploid shrimp was abnormal. Flow cytometry was used to detect the ploidy of sperm in the vas deferens. The data showed that triploidy could affect the sex ratio in Chinese shrimp. The female-to-male ratio in triploids of about 4:1 will favor triploid shrimp aquaculture.

Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis

Chromosome manipulation for commercially valuable marine animals plays an important role in aquaculture. The special reproductive characteristics of shrimp make it difficult to control fertilization and synchronize egg development, so research on chromosome manipulation in shrimp has proceeded very slowly. In the present study, triploid shrimp Fenneropenaeus chinensis were induced by heat shocks and the optimal-inducing condition was screened at different spawning temperatures. Level of triploid induction for each treatment was evaluated by flow cytometry at nauplius stage. The highest level of triploid induction reached to more than 90%. Starting time for each treatment was very crucial for triploid induction in shrimp. One optimal treatment condition for triploid induction was heat shock (29-32 degreesC), starting at 18-20 min for duration of 10 min. These conditions varied depending on the temperature at spawning. Triploid level at embryo stage and nauplius stage was not different, suggesting the same hatching rate between diploids and triploids. Heat shock is a very effective way to induce triploids in this species, and can be easily used on large scale without any harmful effect on the environment as compared with chemical treatment. (C) 2003 Elsevier Science B.V. All rights reserved.

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes-GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.