Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei

A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection.     

Purification and characterization of alpha 2-macroglobulin from the white shrimp (Penaeus vannamei)

alpha(2)-Macroglobulin (alpha(2)M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp alpha(2)M is a 380 kDa protein, a homodimer of two apparently identical subunits of approximately 180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus alpha(2)M counterpart. The shrimp alpha(2)M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp alpha(2)M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by alpha(2)M was provided by inactivation with methylamine.     

Penaeus vannamei isotrypsins: purification and characterization

Three isotrypsins from digestive gland of Penaeus vannamei were purified and characterized by molecular, biochemical and kinetic parameters. Purified isotrypsins A, B, and C are glycoproteins with molecular masses between 30.2 and 32.9 kDa, and, therefore similar to other trypsins. The isoelectric points are anionic and different among the three isotrypsins: pH 3.5 for isotrypsin A, pH 3.0 for isotrypsin B, and pH 4.5 for isotrypsin C. Differences in the NH(2)-terminal amino acid sequences allowed us to define three different protein entities that match isotrypsins previously deduced by cDNA. Isoform C has higher physiological efficiency and specific activity, lower K(m), and requires higher concentrations of Ca(+2) to reach the same activity as the other two isotrypsins.     

Molecular characterization of vitellin from the ovaries of the white shrimp Penaeus (Litopenaeus) vannamei

Vitellin (Vt) was purified from ovary extracts of mature females of the white shrimp Penaeus vannamei using Sepharose CL-4B and Q-Sepharose columns. Native Vt had an apparent molecular weight of 388 kDa as detected in Native-PAGE, bound the lipophilic dye Oil Red O and had a total lipid content of approximately 43.8%. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of three major subunits of 87, 78 and 46 kDa, although minor bands of 65, 61 and 31 kDa are also detected. The 87- and 78-kDa polypeptides were strongly recognized by Penaeus semisulcatus anti-Vt polyclonal and Penaeus monodon anti-Vt monoclonal antibodies. Furthermore, the N-terminal amino acid sequence of the 78-kDa polypeptide is very similar to Penaeus japonicus vitellogenin (Vg) and P. semisulcatus Vt, with an identity of 76%. Circular dichroism indicates that the beta-helix content of Vt is 25% while beta-sheets correspond to 37 and 14% of unordered secondary structure. These values are similar to insect microvitellogenin. Vt has an emission fluorescence maximum at 329 nm, comparable to the shrimp high-density lipoprotein/beta-glucan binding protein (HDL/BGBP).     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Inhibition kinetics of hydrogen peroxide on beta-n-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)

The effects of hydrogen peroxide (H2O2) on prawn NAGase activity for the hydrolysis of pNP-beta-D-GlcNAc have been studied. The results show that H2O2 can reversible inhibit the enzyme (IC50 = 0.85 M) and the inhibition is of a mixed type. The kinetics show that k+o is much larger than k+0, indicating the free enzyme is more susceptible than the enzyme-substrate complex in the H2O2 solution. It is suggested that the presence of the substrate offers marked protection against inhibition by H202. Changes of activity and conformation of the enzyme in different concentrations of H202 have been compared by measuring the fluorescence spectra and residual activity and show that the change of conformation is more rapidly than that of the residual activity, which implies that the whole conformation of the enzyme changes more rapidly than the conformation of the active centre of the enzyme in the H2O2 solution.     

Inhibitory kinetics of bromacetic acid on beta-N-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)

beta-N-Acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52), a composition of chitinases, cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine (NAG). NAGase from prawn (Penaeus vannamei) is involved in digestion and molting processes. The investigation of enzymatic properties, functional groups and catalytic mechanism is an essential mission to its commercial application. Bromacetic acid (BrAc) is a specific modifier for the histidine residue in specific condition. In this paper, the effect of BrAc on prawn NAGase activity for the hydrolysis of pNP-NAG has been investigated. The results showed that BrAc can reversibly and non-competitively inhibit the enzyme activity at appropriate concentrations and the value of IC(50) was estimated to be 17.05+/-0.65 mM. The inhibition kinetics of the enzyme by BrAc has been studied using the kinetic method of the substrate reaction. And the inhibition model was set up and the microscopic rate constants for the reaction of the inhibitor with free enzyme and the enzyme-substrate complexes were determined for inactivation and reactivation. The rate constant of the forward inactivation (k(+0)), which is 1.25 x 10(-3)s(-1), is about eight times as much as that of the reverse reactivation (k(-0)), which is 1.64 x 10(-4)s(-1). Therefore, when the BrAc concentration is sufficiently large, the enzyme is completely inactivated.     

Molecular characterization of the bifunctional VHDL-CP from the hemolymph of white shrimp Penaeus vannamei

A very high-density lipoprotein (VHDL) purified from the hemolymph of the white shrimp Penaeus vannamei is shown to be identical to the clotting protein (CP) previously reported from the same organism based on size, subunits and N-terminal amino acid sequence. The approximately 440-kDa protein, a homodimer of approximately 200-kDa subunits, was present in KBr gradient fractions ranging in density from 1.155 to 1.212 g/ml. Samples of VHDL after purification by strong cation exchange chromatography were subjected to electrophoresis on native polyacrylamide gels. Lipids associated with the VHDL were detected by Sudan Black and Oil Red O staining and comprise 9-15% of the purified protein. Circular dichroism of VHDL-CP indicates that the alpha-helix content of the VHDL-CP is 32%, while beta-sheets correspond to 33%, closely resembling the secondary structure of CP from the shrimp Penaeus monodon and, remarkably, the secondary structure of very high-density lipophorin E (VHDLpE) from the tobacco hornworm, Manduca sexta.     

Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5′-UTR, a coding sequence of 1515 bp and a 104-bp 3′-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (55 kDa) and the tetrameric protein (230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.     

南美白对虾体壁几丁质酶的理化特性研究

报道南美白对虾体壁几丁质酶(EC3.2.1.14)的理化性质。结果表明,酶的最适pH值为5.6,最适温度为55℃。该酶在pH5.0～6.2区域较稳定,而在pH>7和pH<4.6下失活加快;在50℃以下处理1h,酶活力保持稳定,高于55℃,酶稳定性较差,很快失活。研究金属离子对酶活力影响,结果表明:Li 、Na 、K 、Mg2 和Fe2 等对该酶活力没有任何效应;Ca2 、Ba2 对酶有激活作用,Cu2 、Co2 对酶的效应先表现为激活后转为抑制作用;Ni2 、Zn2 、Mn2 、Al3 、Fe3 、Hg2 、Pb2 和Cd2 对该酶活力均具有不同程度的抑制作用,以Hg2 的抑制作用最显著,10mmol/L的Hg2 可抑制酶活力95.6%。     

Characterisation of a serine proteinase from Penaeus vannamei haemocytes

Serine proteinases are involved, besides digestive role, in immune response processes. In addition to the typical serine proteinase domain, proteinases from arthropod haemocytes contain so-called clip domains which are believed to exert regulatory functions. Clones coding for clip domain-containing serine proteinases were isolated from both Penaeus vannamei and Penaeus monodon haemocyte cDNA libraries. These proteins have most of the structural characteristics of serine proteinase domain, but in the clip domain there are only four cysteines, whereas in most other clip domains there are six. Such structures are named pseudo-clip domains and apparently seem to be widely distributed in Penaeid shrimp. These proteinases were only expressed in haemocytes and not in muscles, hypodermis, heart, tail stalk, pleopods or hepatopancreas.     

Proteinase activity in the white shrimp (Penaeus vannamei) clotting protein

The clottable protein (CP) was isolated from white shrimp, Penaeus vannamei plasma as a 400-kDa protein that splits to two identical 200-kDa subunits when it is reduced with 2-ME. However, using DTT as reducing agent, four main bands were observed; two of them (179 and 125 kDa) had the same N-terminus sequence of the intact CP, indicating that most fragmentation occurs in the carboxy-terminus. The proteinase activity of reduced CP was detected using azoalbumin as substrate. Proteinase activity was only detected in the reduced, but not alkylated protein. Trypsin and papain, as well as soybean trypsin inhibitor and E64, were included for comparison. Proteolytic activity of reduced CP was inhibited by E64, but not by STI, indicating that such activity corresponds to a cysteine type proteinase.     

Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon

A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.     

Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei

Mortalities of Penaeus vannamei, cultured in ponds in Belize, Central America, began during the last part of the grow-out cycle during the cold weather months from September 2004 through February 2005. Tissue squashes of infected hepatopancreata and histological examination of infected shrimp revealed that the mortalities might have been caused by an endoparasite. To confirm the diagnosis, DNA was extracted from ethanol preserved hepatopancreata and the small-subunit rRNA gene was sequenced. The 1838 bp sequence was novel and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as a sister taxon to a clade that includes Bonamia and Minchinia species. In situ hybridization was performed using anti-sense DNA probes that were designed to hybridize specifically with the parasite's nucleic acid. This organism presents similar characteristics to those of a haplosporidian that infected cultured P. vannamei imported from Nicaragua into Cuba, as described by Dyková et al. (1988; Fish Dis 11:15-22).     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Purification, biochemical characterization and N-terminal sequence of a serine-protease with chymotrypsic and collagenolytic activities in a tropical shrimp, Penaeus vannamei (Crustacea, Decapoda).

1. Two chymotrypsin variants, with collagenolytic activities, were purified from the hepatopancreas of Penaeus vannamei using radioactive protein as the substrate. 2. These proteases are very close as far as amino acid composition, molecular weight, inhibitors studies and specificity against small synthetic substrates are concerned. 3. N-terminal amino acid sequences of both variants are identical and are very close to other known crustacean serine proteases.     

Sequence and Conservation of a rRNA and tRNAVal Mitochondrial Gene Fragment from Penaeus californiensis and Comparison with Penaeus vannamei and Penaeus stylirostris

Penaeus californiensis is an important species for shrimp fisheries in the Pacific Ocean and has recently been described as a potential cultured species, mainly through the winter season in subtropical regions. A fragment of the mitochondrial 12S rRNA–tRNAVal–16S rRNA genes from P. californiensis was sequenced and compared with the corresponding regions from Penaeus vannamei and Penaeus stylirostris. Purified mitochondrial DNA was used for polymerase chain reaction amplification with primers for 12S and 16S rRNA genes. A 1379 ± 1-bp fragment was obtained, including 90% 16S rRNA, tRNAVal, and a portion of 12S rRNA, cloned, and sequenced. Genetic distances were calculated according to the Kimura 2-parameter distance model, and maximum-likelihood analysis was applied with 1000 bootstrap replications. Sequence identity of P. californiensis with both P. vannamei and P. stylirostris was 0.88, while for P. vannamei and P. stylirostris the identity was 0.92. Maximum-likelihood analysis grouped P. vannamei and P. stylirostris separately from P. californiensis.     

Lecithin essay in the diet of young Penaeus vannamei (Crustacea: Decapoda)

The effect of different lecithin sources and presentations on growth, food conversion ratio and survival of P. vannamei (290 mg +/- 0.02) was studied. The bioassay was designed in order to compare different dietary levels and different quality of lecithin. Squid lecithin, crude soybean (7%), deoiled soybean lecithin (3.48%) in combination with fish oil or squid neutral lipids, in a partially dilapidate formula. The isoenergetic diets were fed ad libitum to four replicate groups (tanks) of 15 shrimps each (5 x 4 x 15), during 28 days. The result of the bioassay with the partially dilapidate formulas was; the best growth rate (191%) and FCR (1.69 +/- 0.041) were obtained with the diet containing 7% of soybean crude lecithin as the unique lipid source. Followed by the diet countering 3.94% deoiled lecithin and 2.42% Menhaden oil (172% and 2.03 +/- 0.054 respectively). As expected, the worst results were obtained without the dietary lecithin 121% and 2.42 +/- 0.129). Crude soybean lecithin alone covered the phospholipid and neutral lipids requirements as well as the combination of deoiled soybean lecithin with fish or squid oil.