Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

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.

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.

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.

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.

Foreign gene transfer into Chinese shrimps (Penaeus chinensis) with gene gun

Plasmids pG DNA-RZ1 with a GFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were observed with a fluorescent microscope. The transient expression of GFP gene was high in nauplius and zoea larvae. Results from RT-PCR and PCR for adults showed that the foreign genes had been transferred into the shrimps and had expressed the corresponding proteins. This work has established a transgenic method for penaeid shrimps, which will set base for the application of genetic engineering breeding into industry.

Morphological and phylogenetic analysis of a Gymnodinium-like species from the Chinese Coast

A Gymnodinium-like species was studied with light microscopy (LM) and scanning electron microscopy (SEM). Also, the internal transcribed spacers (containing 5.8S rDNA) and large ribosomal subunit DNA (D1-D2) sequences were obtained by PCR amplification, and then sequenced to explore the relationships within our isolate, Gymnodinium and other Gymnodinium-like species, including Karenia, Gyrodinium, Karlodinium and Symbiodinium. The LM observation showed that the species was characterized by moving in a levorotatory direction, visible hypocone, epicone and transverse groove, all of which are typical for Gymnodinium. In addition, two flagella could be found under SEM. The phylogenetic analysis revealed that the isolate grouped with Symbiodium, rather than other relevant dinoflagellates. All results showed our isolate belongs to Symbiodium. The strain was isolated from a red tide water sample, denoting that Symbiodium may be causative species for algal bloom.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

cDNA cloning, characterization and mRNA expression of a pacifastin light chain gene from the Chinese mitten crab Eriocheir sinensis

The pacifastin family, characterized by several conserved arrays of six cysteine residues, is a newly identified serine protease inhibitor (SPI) family discovered uniquely in arthropods and plays important roles in multiple biological processes. In the present study, the full-length cDNA of a pacifastin light chain (designated ESPLC) was cloned from the Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1036 bp ESPLC cDNA contained an 831 bp open reading frame (ORF) encoding a putative pacifastin-related peptide of 276 amino acids, a 5'-untranslated region (UTR) of 67 bp, and a 3'-UTR of 138 bp. Six putative conserved domains sharing a characteristic cysteine array (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys) were identified in the deduced amino acid sequence of ESPLC. The conservation of these PLDs (pacifastin light chain domains) and the relative higher similarity of ESPLC to other pacifastin-related precursors suggested that ESPLC was a member of pacifastin family. The mRNA transcripts of ESPLC were found to be higher expressed in hepatopancreas, gill and haemolymph than in gonad, muscle and heart, with the highest expression level in hepatopancreas. The ESPLC mRNA expression in haemolymph of Chinese mitten crab was up-regulated at 2 h and 12 h after challenged with Listonella anguillarum. The tissue distribution and temporal characteristics of ESPLC mRNA expression, similar to that of prophenoloxidase gene in E. sinensis, suggested that ESPLC was potentially involved in the response against invading bacteria, with the possibility that it functioned in the prophenoloxidase system in E sinensis. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved.