Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Adjuvant effects on protection and immune response of Japanese flounder immunized by the formalin-killed cells of Edwardsiella tarda.

We evaluated the effects of Freund's adjuvants (FCA/FIA) on protection and immune response of Japanese flounder Paralichthys olivaceus immunized by the formalin-killed cell (FKC) of Edwardsiella tarda. Combination of FKC and FCA/FIA did not confer protection against the challenge, while they significantly induced higher antibody titers than that of FKC only. The suppression of FKC-dependent induction of interferon γ (IFNγ) mRNA levels by FCA/FIA was observed by gene expression profiling. Similarly, interleukin (IL)-12 p35 mRNA levels were not detected after FKC+FCA or +FIA. These results suggest that the mineral oil in Freund's adjuvants might suppress the signaling pathway(s) that induce IFNγ and IL-12 gene expression.

Effects of 5-Aminolevulinic Acid on Gene Expression, Immunity, and ATP Levels in Pacific White Shrimp, Litopenaeus vannamei.

With the emergence of several infectious diseases in shrimp aquaculture, there is a growing interest in the use of feed additives to enhance shrimp immunity. Recently, the use of 5-aminolevulinic acid (5-ALA), a non-protein amino acid that plays a rate-limiting role in heme biosynthesis, has received attention for its positive effect on immunity in livestock animals. To evaluate the effect of 5-ALA in the Pacific white shrimp, Litopenaeus vannamei, we conducted microarray analysis, a Vibrio parahaemolyticus immersion challenge test, an ATP level assay, and gene expression analysis of some hemoproteins and genes associated with heme synthesis and degradation. Out of 15,745 L. vannamei putative genes on the microarray, 101 genes were differentially expressed by more than fourfold (p < 0.05) between 5-ALA-supplemented and control shrimp hepatopancreas. 5-ALA upregulated 99 of the 101 genes, 41 of which were immune- and defense-related genes based on sequence homology. Compared to the control, the 5-ALA-supplemented group had a higher survival rate in the challenge test, higher transcript levels of porphobilinogen synthase, ferrochelatase, catalase, nuclear receptor E75, and heme oxygenase-1 and higher levels of ATP. These findings suggest that dietary 5-ALA enhanced the immune response of L. vannamei to V. parahaemolyticus, upregulated immune- and defense-related genes, and enhanced aerobic energy metabolism, respectively. Further studies are needed to elucidate the extent of 5-ALA use in shrimp culture.

Identification of novel copper/zinc superoxide dismutase (Cu/ZnSOD) genes in kuruma shrimp Marsupenaeus japonicus.

Superoxide dismutases (SODs) protect cells from superoxides, but in invertebrates, they also have role in the innate immune system. In this study, the genes for five isoforms of copper/zinc superoxide dismutase (MjCu/ZnSOD) gene were identified and sequenced in kuruma shrimp, Marsupenaeus japonicus. The coding parts of the genes ranged from 516 to 585 bp in length and encoded from 172 to 194 amino acids. Structure, phylogenetic and BLAST analyses indicated that MjCu/ZnSOD isoform_3 and _5 belonged to extracellular Cu/ZnSOD (ecSOD) group while the other three isoforms belong to the intracellular Cu/ZnSOD family. In healthy shrimp, the highest expressions of isoform 2, 3 and 4 were in the gills, whereas the expression of isoform 5 was highest in hemocytes. Challenging the shrimp with WSSV and Vibrio penaeicida up-regulated the mRNA expressions of isoforms 3 and 5, suggesting that these isoforms have roles in the innate immune system of kuruma shrimp.

Microarray analysis of hepatic gene expression in juvenile Japanese flounder Paralichthys olivaceus fed diets supplemented with fish or vegetable oils.

Gene expression profiling was performed in Japanese flounder Paralichthys olivaceus fed diets supplemented with fish oil (FO), linseed oil (LO), or olive oil (OO) for 6 weeks. The LO and OO groups showed significantly retarded growth, lower feed intake, lower protein efficiency ratio, and lower hepatosomatic index (P < 0.05). Liver fatty acid composition reflected the dietary fatty acid composition. Microarray analysis revealed that dietary n - 3 highly unsaturated fatty acid (HUFA) deficiency affected 169 transcripts. In the LO group, 57 genes were up-regulated and 38 genes were down-regulated, whereas in the OO group nine genes were up-regulated and 87 genes were down-regulated. Analysis of the functional annotations suggested that dietary n - 3 HUFA affected genes involved in signal transduction (23.2 %), cellular processes (21.1 %), metabolism (including glucose, lipid, and nucleobase; 15.5 %), transport (11.3 %), regulation of transcription (10.5 %), and immune response (4.2 %). Several genes encoding serine/threonine kinases such as protein kinase C and calmodulin-dependent kinase and nuclear hormone receptors such as vitamin D receptor, retinoic acid receptor, and receptors for cytokines (bone morphogenic protein and transforming growth factor ß) were affected. Among 169 transcripts, 22 genes were affected in both LO and OO groups. The present study identified several genes involved in n - 3 HUFA deficiency-sensitive pathways, which will be useful for selective breeding of flounder strains able to adapt to n - 3 HUFA deficiency.

Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster.

BACKGROUND: Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates. PRINCIPAL FINDINGS: We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions. CONCLUSIONS: We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

Effects of ergothioneine from mushrooms (Flammulina velutipes) on melanosis and lipid oxidation of kuruma shrimp (Marsupenaeus japonicus).

The antimelanosic and antioxidative properties of a hot water extract prepared from the fruiting body of the edible mushroom (Flammulina velutipes) were evaluated by dietary supplementation in Kuruma shrimp (Marsupenaeus japonicus) for possible aquaculture application. The extract contained ergothioneine (ERT) at a level of 2.05 mg/mL. A commercial standard of l-ergothioneine (l-ERT) and the mushroom extract showed inhibitory activity against mushroom polyphenoloxidase (PPO). Feeding of the extract had no adverse effects on the immune systems of the shrimp under the present experimental conditions. Supplementation of the extract in the diet significantly suppressed PPO activities in the hemolymphs of the shrimp. Expression of the prophenoloxidase (proPO) gene decreased in the hemocyte of the Kuruma shrimp fed with the mushroom extract. Consequently, development of melanosis was significantly suppressed in the supplement fed shrimp during ice storage. Lipid oxidation was also effectively controlled in the supplement fed group throughout the storage period. In vitro experiments showed that l-ERT effectively inhibited the activation of proPO in the hemocyte lysate supernatant (HLS). The transcript of the proPO gene in the hemocyte showed lower expression in the l-ERT-treated HLS. It was concluded that dietary supplementation of the mushroom extract in shrimp could be a promising approach to control post mortem development of melanosis and lipid oxidation in shrimp muscles.

Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon.

BACKGROUND: White spot syndrome (WSS) is a viral disease that affects most of the commercially important shrimps and causes serious economic losses to the shrimp farming industry worldwide. However, little information is available in terms of the molecular mechanisms of the host-virus interaction. In this study, we used an expressed sequence tag (EST) approach to observe global gene expression changes in white spot syndrome virus (WSSV)-infected postlarvae of Penaeus monodon. RESULTS: Sequencing of the complementary DNA clones of two libraries constructed from normal and WSSV-infected postlarvae produced a total of 15,981 high-quality ESTs. Of these ESTs, 46% were successfully matched against annotated genes in National Center of Biotechnology Information (NCBI) non-redundant (nr) database and 44% were functionally classified using the Gene Ontology (GO) scheme. Comparative EST analyses suggested that, in postlarval shrimp, WSSV infection strongly modulates the gene expression patterns in several organs or tissues, including the hepatopancreas, muscle, eyestalk and cuticle. Our data suggest that several basic cellular metabolic processes are likely to be affected, including oxidative phosphorylation, protein synthesis, the glycolytic pathway, and calcium ion balance. A group of immune-related chitin-binding protein genes is also likely to be strongly up regulated after WSSV infection. A database containing all the sequence data and analysis results is accessible at http://xbio.lifescience.ntu.edu.tw/pm/. CONCLUSION: This study suggests that WSSV infection modulates expression of various kinds of genes. The predicted gene expression pattern changes not only reflect the possible responses of shrimp to the virus infection but also suggest how WSSV subverts cellular functions for virus multiplication. In addition, the ESTs reported in this study provide a rich source for identification of novel genes in shrimp.

Cloning, expression and functional analysis of a novel-chemokine gene of Japanese flounder, Paralichthys olivaceus, containing two additional cysteines and an extra fourth exon.

A CC chemokine gene (JFCCL3) was cloned and sequenced from Japanese flounder, Paralichthys olivaceus. The JFCCL3 cDNA contains an open reading frame of 288 nucleotides encoding 95 amino acid residues. The predicted amino acid sequence of JFCCL3 showed the conserved cysteine of the beta chemokine plus two additional cysteines. The genomic sequence consists of two isoforms: JFCCL3.1 and JFCCL3.2 with sizes of 1.8 and 1.2kb, respectively. Both isoforms contain three introns and four exons. RT-PCR showed that JFCCL3 is constitutively expressed in most tissues including lymphoid organs. Using quantitative real-time RT-PCR, the highest expression of JFCCL3 transcripts was observed in PBLs at 3h post-stimulation with Con A/PMA and at 1h post-stimulation with LPS. A phylogenetic analysis showed that JFCCL3 is more closely related to fractalkines than to other mammalian beta chemokines. A chemotaxis assay showed that recombinant JFCCL3 protein has bioactivity for Japanese flounder leukocyte attraction at concentrations from 0.01 to 10 microg/ml.