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.     

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.     

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.     

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).     

Rickettsial and mollicute infections in hepatopancreatic cells of cultured Pacific white shrimp (Penaeus vannamei)

Infections by multiple species of bacteria occurred in hepatopancreatic epithelial cells of cultured Pacific white shrimp (Penaeus vannamei). Grossly, hepatopancreases of moribund shrimp were pale white. Light microscopically, hepatopancreatic tubules appeared atrophied and were associated with granulomas. Examination by scanning and transmission electron microscopy revealed heavy cytoplasmic infections by three forms of microorganisms: (1) a rickettsia-like bacterium, (2) a helical form of a mollicute-like bacterium, and (3) a filamentous mollicute-like bacterium. The rod-shaped rickettsia (900 nm long by 300 nm wide) appeared to be free in the cytoplasm and had both a plasma membrane and a cell wall. Neither form of mollicute possessed a cell wall. The helical mollicute was blunt at its wide end (about 260 nm in diameter) where it contained electron-lucent bodies. Helical turns along its tapered axis resembled those of a spiroplasma (the only helical form of mycoplasma in the class Mollicutes) or a spirochete. The helical bacterium did not possess periplasmic flagella characteristic of spirochetes, which lends support to its being a type of spiroplasma. The filamentous mollicute consisted of masses of short, branched filaments 60 nm wide with intermittent spherical dilations and terminal blebs on the branches. The presumed mollicutes have not been reported previously in crustaceans. Each bacterium, or concurrent infections of the bacteria, are pathogenic to cultured shrimp, could impact culture operations and thus deserve more study.     

1,3-beta-D glucan binding protein (BGBP) from the white shrimp,Penaeus vannamei,is also a heparin binding protein

Shrimp BGBP was purified as a 100 kDa glycoprotein by affinity chromatography using immobilised heparin. BGBP bound simple carbohydrates, glycosaminoglycans like heparin sulphate and glycoproteins, but it was unable to agglutinate erythrocytes. Using an ELISA-based microplate assay, it was shown that simple carbohydrates such as n-glucose and D-mannose are competitive inhibitors of heparin sulphate binding to BGBP. Based on these properties BGBP is considered as a new type of heparin binding protein.     

Metabolic responses of the white shrimp, Penaeus vannamei, to ambient ammonia

Juvenile shrimp were individually exposed during 24 h to 0.007 (control), 0.36, 1.07, and 2.14 mmol/l total ammonia-N at 28 degrees C and 39 ppt salinity. After 22 h of ammonia-N exposure, oxygen consumption was measured for 2 h, and then hemolymph, hepatopancreas, and muscle tissues were sampled. Oxygen consumption, and levels of lactate and glycogen in the hepatopancreas increased significantly at the highest ammonia-N concentration. Concentration of oxyhemocyanin, acylglycerol, and cholesterol in hemolymph, and lactate in muscle decreased significantly in the group exposed to the highest ammonia levels. The changes observed in hemolymph and tissue metabolic fuels suggest a reduced use of carbohydrate through anaerobic metabolism and an increase in the use of lipids to satisfy the metabolic demand.     

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.     

Pyrokinin neuropeptides in a crustacean. Isolation and identification in the white shrimp Penaeus vannamei.

Identification of substances able to elicit physiological or behavioural processes that are related to reproduction would greatly contribute to the domestication of commercially important crustaceans that do not reproduce easily in captivity. Crustaceans are thought to release urine signals used for chemical communication involved in courtship behaviour. In contrast to insects, very little is known about the endocrinological processes underlying this phenomenon. Therefore, an extract of 3500 central nervous systems of female white shrimp Penaeus vannamei was screened for myotropic activity in order to purify pyrokinin-like peptides that belong to the pyrokinin/PBAN neuropeptide family. Members of this family regulate reproductive processes in insects, including pheromone biosynthesis. Purification of these pyrokinins was achieved by a combination of reversed-phase and normal-phase chromatography. Subsequent characterization by mass spectrometry, Edman degradation and peptide synthesis resulted in the elucidation of two novel peptides. Pev-PK 1 has the primary sequence DFAFSPRL-NH(2) and a second peptide (Pev-PK 2) is characterized as the nonapeptide ADFAFNPRL-NH(2). Pev-PK 1 contains the typical FXPRL-NH(2) (X = G, S, T or V) C-terminal sequence that characterizes members of the versatile pyrokinin/PBAN family. Pev-PK 2 displays an Asn residue at the variable X position of the core pyrokinin sequence. These crustacean pyrokinins are the first to be found in a noninsect. The synthetic peptides display myotropic activity on the Leucophaea maderae as well as on the Astacus leptodactylus hindgut.     

Reconsideration of phenoloxidase activity determination in white shrimp Litopenaeus vannamei

Though phenoloxidase (PO) activity has been used as an important index in immunological research of crustaceans, methods for the determination of PO activity are not consistent even for the same species. Plasma, the major location of PO activity, should be the most reasonable sample, instead of hemocytes or serum, for the determination of PO activity of shrimp. The current study provided a thorough characterization and reconsideration for PO activity assay in the plasma of Litopenaeus vannamei. Results show that the final concentration of l-dihydroxyphenylalanine (l-DOPA) for PO activity assay should be no less than 1.5 mg ml^?1, and pH 6.6 should be used to maintain the stability of l-DOPA solution. This study provides direct evidence that PO activity is significantly inhibited by EDTA, and it is suggested to use EDTA-free anticoagulant in separating plasma for PO activity assay in future studies. Repeated measurements indicated that the assayed PO activities are significantly affected by preservation conditions, and plasma is quite unstable with spontaneous activation when put in ice or stored at ?20 °C. Thus samples need to be measured immediately or preserved at ?80 °C with assay as soon as possible after it is thawed, and should not be preserved for a second time for measuring PO activity.     

Yolk synthesis in the marine shrimp,Penaeus vannamei

• 1.1. In vitro yolk synthesis was measured in fragments of the ovary of developing shrimp, Penaeus vannamei.
• 2.2. Progesterone and estradiol stimulated yolk synthesis in vitro, while ecdysterone, testosterone and estrogen had no effect.
• 3.3. A peptide factor from the eyestalks of crayfish stimulated yolk synthesis in vitro. A peptide factor from shrimp eyestalks inhibited yolk synthesis in vitro.

     

Experimental evidence of metabolic disturbance in the white shrimp Penaeus vannamei induced by the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV)

Xenorhabdus budapestensis can produce a variety of proteins that help this bacterium and its mutualistic nematode vector kill the host insect. In this report, we purified one protein fraction from the intracellular extract of X. budapestensis D43, which was designated HIP57. By injection, HIP57 caused Galleria mellonella larval bodies to blacken and die with an LD(50) of 206.81 ng/larva. Analyzes of HIP57 by two-dimensional gel electrophoresis showed that this protein was a single spot on the gel with a molecular weight of 57 kDa and a pI of ～5. Sequencing and bioinformatic analysis suggested that the HIP57 toxin was homologous to GroEL. GroEL has been accepted as molecule chaperon; however, our research revealed that HIP57 (GroEL) possesses another novel function as an insecticide. A GroEL phylogenetic tree defined the relationship among the related species of mutualistic bacteria (Xenorhabdus and Photorhabdus) from the entomopathogenic nematodes and the evolution within the family Enterobacteriaceae. Thus, GroEL could be a complement to 16S rDNA for studying the molecular phylogenies of the family Enterobacteriaceae. Phenoloxidase (PO) activity analysis of G. mellonella larvae injected with HIP57 suggested that the toxin activates the PO cascade, which provides an extensive defense reaction that potentially responsible for G. mellonella larval death.     

Seaweed meal as a protein source for the white shrimp Litopenaeus vannamei

The rhodophytes Hypnea cervicornis and Cryptonemia crenulata are abundant along the Brazilian coastline and are rich in nutrients. They may therefore be used as a source of protein in shrimp diets. The aim of the present study was to test this hypothesis. The experiment was conducted in a laboratory, where 10-day-old post-larvae aged underwent 7 days of acclimation in a 1,000 L tank. They were then kept in plastic aquariums, each containing 10 L, and 20 larvae were fed daily (10% of biomass) in four equal portions with one of four diets (five repetitions of each) for a period of 45 days. All diets contained 30% crude protein (isoprotein) and 300 kcal 100 g⁻¹ (isocaloric), with different percentages of seaweed powder: Diet “A” 39%; Diet “B” 26%, Diet “C” 13%, and Diet “D” without seaweed (control diet). Algae were collected, rinsed, dried and ground up for the feed formulations. Weight of the animals was measured at the beginning of the experiment and at 15-day intervals to assess their growth. The physico-chemical variables of the water were measured every 2 days. Final biomass, biomass gain and specific growth rate (SGR) exhibited no significant differences between treatments (P > 0.05). Survival rate was equal under the four experimental conditions, being consistent within four decimal places 95.2% to 97.00% (P > 0.05). Diets “A” and “B”, with a greater content of algae, exhibited better feed conversion (1.79:1 and 1.82:1) than Diets “C” and “D” (2.04:1 and 2.08:1) (P < 0.05). The physical-chemical variables of the water showed no significant variation and remained within the standards necessary for the wellbeing of the animals. If sufficient biomass of beached algae can be practically and economically collected, it may be used as a component in the making of shrimp feed.     

Vaccination of shrimp (Penaeus chinensis) against white spot syndrome virus (WSSV)

Two structural protein genes, VP19 and VP466, of white spot syndrome virus (WSSV) were cloned and expressed in Sf21 insect cells using a baculovirus expression system for the development of injection and oral feeding vaccines against WSSV for shrimps. The cumulative mortalities of the shrimps vaccinated by the injection of rVP19 and rVP466 at 15 days after the challenge with WSSV were 50.2% and 51.8%, respectively. For the vaccination by oral feeding of rVP19 and rVP466, the cumulative mortalities were 49.2% and 89.2%, respectively. These results show that protection against WSSV can be generated in the shrimp, using the viral structural protein as a protein vaccine.     

Immunostimulation of white shrimp (Litopenaeus vannamei) following dietary administration of Ergosan

Ergosan an algal product containing 1% alginic acid, developed for use in aquaculture and reported to have immunomodulatory activity, was administered orally to intermoult adult white shrimp (Litopenaeus vannamei) for 15 days. Examination of haemolymph proteins using SDS-PAGE did not reveal any obvious differences between control and Ergosan treated shrimp. Similarly, total haemocyte counts were found to be roughly equivalent for both the control and experimental samples. However, differential analysis of haemocyte populations revealed marked changes in terms of the relative levels of hyaline, semi-granular, and particularly granular haemocytes between the two groups. Moreover, enhancement of the in vitro antimicrobial activity of haemolymph towards two shrimp pathogenic Vibrio isolates was recorded for shrimp fed with Ergosan. Finally, shrimp fed with Ergosan showed a significant increase in relative growth when compared with control groups.