Extracellular ATP is a potent signaling molecule in the activation of the Japanese flounder (Paralichthys olivaceus) innate immune responses

Innate immunity is the first line of defense against pathogen infections. Extracellular ATP (eATP) is one of the most studied danger-associated molecular pattern molecules that can activate host innate immune responses through binding with and activating purinergic receptors on the plasma membrane. The detailed actions of eATP on fish innate immunity, however, remain poorly understood. In this study, we investigated bacterial pathogen-induced ATP release in head kidney cells of the Japanese flounder Paralichthys olivaceus. We also examined the actions of eATP on pro-inflammatory cytokine and immune-related gene expression, the activity of induced NO synthase (iNOS), and the production of reactive oxygen species (ROS) and NO in Japanese flounder immune cells. We demonstrate that ATP is dynamically released from Japanese flounder head kidney cells into the extracellular milieu during immune challenge by formalin-inactivated Edwardsiella tarda and Vibrio anguillarum. In addition, we show that eATP administration results in profound up-regulation of pro-inflammatory cytokine gene expression, iNOS activity, and inflammatory mediator production, including ROS and NO, in Japanese flounder immune cells. Altogether, our findings demonstrate that eATP is a potent signaling molecule for the activation of innate immune responses in fish.     

Molecular characterization and gene expression of a CXC chemokine gene from Japanese flounder Paralichthys olivaceus

Chemokines are small, secreted cytokine peptides that have the ability to recruit a wide range of immune cells to sites of infection and disease. A novel CXC chemokine was obtained from Japanese flounder Paralichthys olivaceus. This chemokine cDNA contains an open reading frame of 333 nucleotides encoding 111 amino acid residues containing four conserved cysteine residues. The gene is composed of four exons and three introns as are those of mammalian and fish CXC chemokines. Results of homology and phylogenetic analysis revealed that the Japanese flounder CXC chemokine is closest to CXCL13 subgroup. The gene was expressed in immune-related organs, including head kidney, trunk kidney, spleen and peripheral blood leukocytes (PBLs). Japanese flounder CXC chemokine gene expression was observed at 3 and 6h after induction by LPS, but not at 3 and 6h after induction by poly I:C. These results suggest that the Japanese flounder CXC chemokine is probably associated with inflammatory as well as homeostatic functions.     

Molecular cloning of stanniocalcin 1 and its extracorpuscular regulation by salinity and Ca2+ in the Japanese flounder

Stanniocalcin 1 (Stc1) was originally identified as an anti-hypercalcemic hormone produced by the corpuscles of Stannius (CS) associated with the kidney in teleosts. While the stc1 gene is expressed in various tissues in fishes, its role and regulation in extra-CS tissues are unexplored. In the present study, we characterized a cDNA of stc1 in a euryhaline fish, the Japanese flounder (Paralichyhus olivaceus), and examined its expression in peripheral tissues in response to different salinities and Ca2+ ion concentrations. The Japanese flounder stc1 cDNA (1331 bp) encodes a preprohormone of 251 amino acids (aa), with a signal peptide of 17 aa and a pro-sequence peptide of 15 aa followed by the mature protein of 219 aa. The deduced aa sequence of Japanese flounder stc1 showed highest sequence identity (94.0%) with the European flounder Stc1 among fish and mammalian species, but lower identity to zebrafish, pufferfish, and human STC2 (23.1-25.4%). Lowered environmental salinity resulted in a decrease in stc1 mRNA expression in vivo in the gills, kidney, intestine, and CS glands of the Japanese flounder. Furthermore, we found that extracellular Ca2+ increased steady-state stc1 mRNA levels in gill and kidney cells as well as in the CS cells. Our findings suggest that Stc1 synthesis in the ionregulatory tissues is responsive to environmental salinity and Ca2+ level.     

Density-dependent predation and implications for stock enhancement with Japanese flounder

In laboratory predation trials kinsen-gani crab Matuta lunaris rarely preyed on hatchery-reared (HR) Japanese flounder Paralichthys olivaceus , even at extremely elevated prey densities ( c . 90 fish m⁻²), but fed readily on sacrificed Japanese founder. This suggests that under similar conditions in the wild predator-prey interactions between kinsen-gani and juvenile Japanese flounder are likely to be weak. Larger Japanese flounder readily cannibalized smaller conspecifics, suggesting that within-release group cannibalism may contribute considerably to post-release mortality of HR Japanese flounder when sufficient size variation exists within groups of released fish. Kinsen-gani presence had no effect on within-release group cannibalism rates of large Japanese flounder on small Japanese flounder. Within-release group cannibalism rates were significantly affected by densities of smaller (prey) fish, implying that the number of fish released (and thus post-release densities) in stock enhancement programmes may significantly affect post-release mortality rates. The probability of within-release group cannibalism increased rapidly as the cannibal-to-prey size ratio exceeded 1.6, suggesting that minimizing size variation of released HR individuals will reduce post-release cannibalism rates. These results have implications for Japanese flounder stock enhancement release strategies, and may be applicable to other finfish stock enhancement programmes.     

Predator-prey interaction between hatchery-reared Japanese flounder juvenile, Paralichthysolivaceus, and sandy shore crab, Matutalunaris: daily rhythms, anti-predator conditioning and starvation

Predator-prey interaction between sandy shore crab, Matuta lunaris (Forskål, 1775), and juvenile Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel), was investigated under controlled laboratory conditions. Possibility of training and conditioning hatchery-reared flounder to avoid predators was also examined. Crabs took over 75% of their daily ration at night when they were given access to prey 24 h a day. Large (64.8±5.4 g)- and medium (30.68±3.33 g)-sized crabs ate ca. 5.5±1.45 and 3.9±1.99 individuals of flounder (TL=4.96±0.23 cm) a day, respectively. When flounder juveniles that have experienced predation pressure by crabs encountered predators again, they exhibited better survival compared to the naive fish. Flounder juveniles were also conditioned either using small and, thus, benign predators, or large crabs over fence. The conditioned fish with either method were better able to avoid capture by crabs than naive fish, revealing that learning process should play an important role in their predator avoidance. Anti-predator performance was also compared between starved and fed flounder juveniles. Fed fish were rarely eaten by predators after 3 h of exposure, whereas starved fish continued to be eaten. Our results suggest that stock-enhancement program of Japanese flounder can be improved by applying proper feeding protocol and conditioning to avoid predators prior to release. Present research supports the idea that behavioural and ecological consideration for the target species is indispensable for the success of stock enhancement.     

The granulocyte colony-stimulating factors (CSF3s) of fish and chicken

Granulocyte colony-stimulating factor (CSF3) is a glycoprotein cytokine, which influences the hematopoiesis of the phagocytic neutrophils and its precursors and was used extensively in cancer therapy and for the treatment of neutropenia in mammals. However, CSF3 is yet to be identified in nonmammalian species mainly because of its rapid mutation. Here, we report the first CSF3 genes from three teleost fishes: Japanese flounder (Paralichthys olivaceus), fugu (Takifugu rubripes), and green-spotted pufferfish (Tetraodon nigroviridis) and present evidence that the chicken (Gallus gallus) myelomonocytic growth factor is in fact the chicken CSF3 orthologue. We support this by showing significant conservation of the CSF3 genes’ structure, domains, regulatory motifs, and synteny across species and by phylogenetic analysis. CSF3 orthologues are indeed evolving rapidly and appears to be undergoing purifying selection in mammals but positive selection in fish and chicken. Furthermore, the paralogous fugu and pufferfish CSF3-1s and CSF3-2s are shown to be the ancestral and duplicate genes, respectively. Finally, we demonstrate that the Japanese flounder CSF3 gene is at least involved in immunity based on its basal expression in immune-related tissues and its upregulation in kidney and peripheral blood leukocytes after in vitro stimulation with lipopolysaccharide and a combination of concanavalin A/phorbol myristate acetate.Electronic Supplementary Material Supplementary material is available for this article at ()     

Alpha melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1

The ability of interleukin 1 (IL 1) to augment the proliferation of murine thymocytes in vitro was inhibited in a dose-dependent manner by the neuropeptide alpha-melanocyte-stimulating hormone (alpha MSH). The minimal effective concentration of alpha MSH was 10(-11) M. Maximal effect occurred between 10(-8) and 10(-7) M, with diminishing effectiveness at higher concentrations. IL 1-induced production of prostaglandin E (PGE) by fibroblasts was also inhibited by alpha MSH with a biphasic dose response. The minimal effective concentration was 10(-11) M, and maximum effect was achieved at 10(-10) M. alpha MSH appeared to affect the interaction of IL 1 with its target cells in a specific manner, because it did not inhibit basal mitogen-induced thymocyte proliferation or IL 2-induced proliferation of a cytotoxic T lymphocyte line. Furthermore, production of IL 1 by endotoxin-stimulated monocytes was not affected by alpha MSH. An analog of alpha MSH (Nle4, D-Phe7 alpha MSH), which is highly potent in other melanotropin-sensitive systems, did not affect the action of IL 1 on thymocytes, suggesting that the immunomodulatory effects of alpha MSH may not be mediated by the classic melanocyte alpha MSH receptor. The influence of alpha MSH on thymocytes and fibroblasts suggests that alpha MSH is an endogenous antagonist of IL 1, perhaps important for limiting inflammatory damage to host tissues.     

Field evaluation by RNA/DNA ratios on post-release nutritional status of released and wild Japanese flounder Paralichthys olivaceus juveniles

A large-scale juvenile Japanese flounder (Paralichthys olivaceus) release-recapture experiment was undertaken to find the optimal release season by evaluating the nutritional status of released fish at different seasons during which food abundance was significantly different. Forty thousand fish were released at depths of 1.5 m for early-release (May 29, 1997) and 2 m for late-release (July 2, 1997) (42.1±3.5 and 40.9±4.2 mm body length, respectively) in an experimental field, Wakasa Bay, the Sea of Japan. Samples were taken, after the releases, at Wada beach at intervals of 1, 2, 3, 6, 10, 16 and 30 days after release (DAR), including pre-surveys before each release. Released fish recaptured from the two different release groups totaled 764; 467 from the early-release group (ER) and 297 from the late-release group (LR). A total of 1956 wild flounder juveniles were simultaneously collected (1041 ER, 915 LR). ER fish were subject to higher food availability and were exposed to less pressure from predation by smaller wild juvenile flounder. RNA/DNA ratios in ER juveniles were significantly higher than those of LR fish during all samples. Especially, RNA/DNA ratios in ER juveniles were higher than in wild juveniles from 3 to 50 DAR. In the LR group, the nutritional status of juveniles was relatively low in shallower water. These findings corresponded well with feeding incidence examined by coworkers. Mass release of hatchery-reared juveniles apparently reduced RNA/DNA ratio of the wild juveniles right after releasing. The present study showed that earlier release of hatchery-reared juvenile Japanese flounder with higher RNA/DNA ratio could increase the possibilities of survival right after release in the nursery ground, and that RNA/DNA ratio appeared to be a good tool in evaluating nutritional status of released juveniles as well as wild juveniles in Japanese flounder.     

Modulation of the early immune response against viruses by a teleostean interferon regulatory factor-1 (IRF-1)

We investigated the role of a teleostean interferon regulatory factor-1 (IRF-1) in the regulation of the fish immune system using Japanese flounder, Paralichthys olivaceus, as a model. Fish were intramuscularly vaccinated with a recombinant plasmid expressing the Japanese flounder IRF-1 (JF IRF-1) under the control of the cytomegalovirus immediate/early enhancer (CMV) promoter and were sampled at different days post-immunization. Peripheral blood leukocytes (PBLs) obtained from the JF IRF-1-vaccinated fish during the early stages post-vaccination had significantly elevated levels of nitric oxide (NO) and higher acid phosphatase (AP) activity compared with the control groups. Moreover, supernatants of PBLs obtained from the IRF-1-vaccinated fish contained cytokine-like substances as shown by their protective effect against hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV) in two cell lines, hirame natural embryo (HINAE) cell line and epithelial papillosum of cyprini (EPC) cell line. Relative expression of an anti-viral gene, Mx was highest at the 7th day post-vaccination. Co-injection of JF IRF-1 with a DNA vaccine encoding the major capsid protein (MCP) gene of red seabream iridovirus (RSIV) resulted in elevated serum neutralization antibodies but was not significantly different from that in the fish vaccinated with the DNA vaccine alone. These results suggest that the JF IRF-1 modulates the early immune response in fish and is a potential candidate as genetic adjuvant for vaccination.     

Glyceraldehyde-3-phosphate dehydrogenase of Edwardsiella tarda has protective antigenicity against Vibrio anguillarum in Japanese flounder

Edwardsiella tarda glyceraldehyde-3-phosphate dehydrogenase (GAPDH) may be an effective vaccine candidate against infection by E. tarda in Japanese flounder Paralichthys olivaceus. The GAPDH of E. tarda is highly homologous to that of Vibrio cholerae (91%), and therefore E. tarda GAPDH may have protective antigenicity against Vibrio species. In this study, we immunized Japanese flounder with GAPDH of E. tarda and infected the fish with V anguillarum. The result showed that GAPDH prepared from E. tarda protected Japanese flounder effectively in a challenge of V anguillarum. Therefore, E. tarda GAPDH should be considered as a multi-purpose vaccine candidate against several kinds of pathogenic bacteria.     

Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.