Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Enhanced cellular immunity in shrimp (Litopenaeus vannamei) after 'vaccination'

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A secretory leukocyte proteinase inhibitor (SLPI)-like protein from Litopenaeus vannamei haemocytes

A partial clone coding for a two-WAP domain protein was isolated from a Litopenaeus vannamei haemocytes cDNA library. The complete sequence was obtained by RACE, and the full-length cDNA sequence is 0.8 Kb long and encodes for a 116-amino acid protein. The domain composition is similar to the mammalian WFDC5 (WAP four disulfide core) and secretory leukocyte proteinase inhibitor (SLPI). Modifications in expression were determined by real-time PCR, after injection of Vibrio alginolyticus, suggesting its participation in the shrimp immune response. Structural and phylogenetic analyses showed close similarity between shrimp and mammalian SLPI, indicating a probable common ancestor. This is the first report of a mammalian SLPI-like protein in an invertebrate.     

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.     

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.     

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei

In this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288-2503bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.     

Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (18.4 +/- 1.2 g) were injected individually with noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1). For the shrimp that received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1), the THC decreased by 15%, 21% and 32%, phenoloxidase activity decreased by 15%, 31% and 31%, respiratory burst decreased by 13%, 21% and 32%, and SOD activity decreased by 46%, 56% and 55%, respectively, after 2 h. The phagocytic activity and clearance efficiency of shrimp that received noradrenaline at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 4, 4, 8, 24, 16 and 8 h, respectively, in the shrimp that received noradrenaline at either dose. In another experiment, L. vannamei which had received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1) were challenged after 1h by injection with V. alginolyticus at 1.0 x 10(5) colony-forming units (cfu)shrimp(-1) and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received noradrenaline at either dose was significantly higher than that of shrimp that received saline after 4 h, and at the termination of the experiment (48 h after the challenge). It is therefore concluded that noradrenaline administration at 10(-6) mol shrimp(-1) or less causes immune modulation of L. vannamei.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

Antigenic analysis of flaviviruses with monoclonal antibodies against Negishi virus

Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.     

A new type of Kazal proteinase inhibitor related to shrimp Penaeus (Litopenaeus) vannamei immunity

A clone encoding a four-Kazal domain-containing protein was isolated from the hemostats of a Penaeus vannamei cDNA library. The full-length cDNA sequence is 975 bp in length and encodes a 24.4 kDa protein (228 residues). Four Kazal domains, each 43-46 residues in length, were detected in the deduced primary structure. The first, third and fourth domains have the CPLREELPVC, CPAVYDPVC and CPLYVDPVC motifs, respectively, suggesting that they are able to inhibit chymotrypsin and elastase. The mRNA levels of the Kazal protein were modified after the injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. All these characteristics are similar to previously reported shrimp Kazal, however, based on both domain architecture and expression profile following Vibrio stimulation, this protein represents a new type of Kazal inhibitor associated with shrimp immunity.     

Antigenic analysis of testicular cytochromes c using monoclonal antibodies

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.     

Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to beta-glucan and sulphated polysaccharide

Juvenile American white shrimp (Litopenaeus vannamei) were immersed in aerated beta-glucan and sulphated polysaccharide solutions for 1, 3 and 6 h. Superoxide anion and SOD activity in haemocytes and muscle were investigated to evaluate whether beta-glucan and sulphated polysaccharide induce any immunostimulatory activity. Haemocytes and muscle showed different levels of superoxide anion generation and SOD activity (2.0 and 14 times that of control, respectively) when shrimp were immersed for 6 h in aerated sea water containing beta-glucan and sulphated polysaccharide. Total haemocyte count (THC) decreased within the first 24 h after challenge with immunostimulants, but THC and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulphated polysaccharide is capable of generating an increase in the respiratory burst of L. vannamei haemocytes.     

Purification and characterization of an intracellular lipase from pleopods of whiteleg shrimp (Litopenaeus vannamei)

An intracellular lipase present in the whiteleg shrimp Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. Purified intracellular lipase has a molecular mass of 196kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30-40°C at pH 8.0-10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930U mg(-1) and 416.56U mg(-1) was measured using triolein and tristearin at pH 8.0 at 30°C as substrates, respectively. The lipase showed a K(M,app) of 41.03mM and k(cat)/K(M,app) ratio of 4.88 using MUF-butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.     

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).     

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.