A cDNA microarray approach for analyzing transcriptional changes in Penaeus monodon after infection by pathogens

A cDNA microarray comprised of 9990 different ESTs obtained from the Penaeus monodon EST project (http://pmonodon.biotec.or.th) was employed to identify viral (white spot and yellow head viruses) and bacterial (Vibrio harveyi) responsive genes in the hemocytes of P. monodon at 6, 24 and 48 h post-injection (hpi). The number of differentially expressed genes found was highest in shrimps infected with white spot virus (1954 genes) followed by yellow head virus (1136 genes) and V. harveyi (420 genes). Changes in shrimp gene expression were highest at the late infection stage for both viruses, whilst that for V. harveyi induced gene expression was mainly found at the early infection stage, but the repression of genes was mainly found in the mid stage of infection. Shrimp genes specifically upregulated by each particular pathogen are identified and are summarized.     

Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.     

Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis

Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum. After incubation with V. tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium. To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V. tapetis to inhibit adhesion of clam hemocytes to plastic. Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp. pathogenic to bivalves, and that of various V. tapetis isolates were tested. Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte. The greater cytotoxic activity of V. tapetis compared to that of V. splendidus and V. pectenicida suggests a specific pathogenicity of V. tapetis to R. philippinarum hemocytes. Although all V. tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated.     

Bacterial clearance rate and a new differential hemocyte staining method to assess immunostimulant activity in shrimp

New methods were developed to assess immunostimulant efficacy in the black tiger shrimp Penaeus monodon. Test shrimp were fed with 2 or 4 % yeast extract (YE)-coated feed while controls were fed non-coated feed. After 4 wk of feeding, individual shrimp were assessed for total hemocyte counts (THC), the number of granular hemocytes (GH) and rate of bacterial clearance. For hemocyte counts, formalin-fixed hemolymph was stained with 1.2 % Rose Bengal in 50 % ethanol for 20 min at room temperature. Some of this mixture was used for THC with a hemocytometer while some was smeared on a microscope slide and left to dry before counterstaining with hematoxylin for GH counts. By this technique, high quality smears were obtained for accurate differential counts. Bacterial clearance assays were used to assess the sum effect of humoral and cellular defense mechanisms. Vibrio harveyi was injected intramuscularly at 1 x 10(8) cells per shrimp and hemolymph was collected in anticoagulant at 0, 15, 30 and 60 min post-injection for quadruplicate drop counts (20 microl) on TCBS agar. Total hemocyte counts for shrimp fed with 4 % YE were significantly higher (p < 0.05) than those for shrimp fed with non-coated feed. The percentage of granular cells and the rates of bacterial clearance for the YE-fed shrimp were higher than those for shrimp fed the control diet. These 2 methods provide a simple and rapid comparison of shrimp groups for differences in anti-bacterial defense capacity.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Nucleotide sequence of the LuxC gene and the upstream DNA from the bioluminescent system of Vibrio harveyi.

The nucleotide sequence of the luxC gene (1431 bp) and the upstream DNA (1049 bp) of the luminescent bacterium Vibrio harveyi has been determined. The luxC gene can be translated into a polypeptide of 55 kDa in excellent agreement with the molecular mass of the reductase polypeptide required for synthesis of the aldehyde substrate for the bioluminescent reaction. Analyses of codon usage showed a high frequency (1.9%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA, B and D genes. The low G/C content of the luxC gene and upstream DNA (38-39%) compared to that found in the other lux genes of V. harveyi (45%) was primarily due to a stretch of 500 nucleotides with only a 24% G/C content, extending from 200 bp inside lux C to 300 bp upstream. Moreover, an open reading frame did not extend for more than 48 codons between the luxC gene and 600 bp upstream at which point a gene transcribed in the opposite direction started. As the lux system in the luminescent bacterium, V. fischeri, contains a regulatory gene immediately upstream of luxC transcribed in the same direction, these results show that the organization and regulation of the lux genes have diverged in different luminescent bacteria.     

An improved method of cell culture system from eye stalk,hepatopancreas, muscle,ovary, and hemocytes of <Emphasis Type="Italic">Penaeus vannamei</Emphasis>

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO₂. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO₂ supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.     

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.     

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.     

In vivo insect hemocyte destruction by UV-irradiated Chilo iridescent virus

Large inoculum of infective or UV-irradiated Chilo iridescent virus (CIV) caused an early drastic cytopathy of the larval hemocytes of the greater wax moth, Galleria mellonella. Cytopathy of the hemocyte could be detected 2 hr after virus injection at 25°C and most hemocytes were completely destroyed within 8 hr after virus injection. Heat-treated CIV did not produce any cytopathy of Galleria hemocytes. Antiserum to CIV neutralized this cytopathic effect, while that to Tipula iridescent virus did not demonstrate any inhibitory effect. Actinomycin D, mitomycin C, and puromycin did not inhibit hemocyte destruction by UV-irradiated CIV. Galleria larva injected with a large dose of UV-irradiated CIV progressively became stunted with a decrease of body weight and did not regain its hemocyte numbers. Hemocytes infected with CIV were not destroyed by any additive injection of a large dose of UV-irradiated CIV.     

Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis

A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis by sequential anion-exchange chromatography. The shrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase present in shrimp hemocytes. Dansylcadaverine was incorporated into the shrimp CP in the presence of endogenous transglutaminase (hemocyte lysate), confirming that the shrimp purified CP is the substrate for the transglutaminase enzyme. The molecular mass of the CP was determined by gel filtration to be 341 kDa and 170 kDa by SDS-PAGE under reducing conditions. These results suggest that the shrimp CP consists of two identical subunits, covalently linked by disulphide bonds. The amino acid sequence at the N-terminus was 100% identical to that of the penaeids Litopenaeus vannamei and Penaeus monodon and 66% to 80% identical to the CPs of other decapods. This is the first report of a CP characterization in an Atlantic penaeid species. Further studies, including a molecular cloning approach would enable to detect which tissues express the gene of the clotting protein. It would be also useful to understand the mechanism by which the coagulation time is delayed in shrimps under stress conditions.