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.     

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species.     

Expression and promoter activity of endogenous retroviruses in the Olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>)

Olive flounder (Paralichthys olivaceus) is regarded as one of economical fish species in the world. Genome information of Olive flounder has been revealed by next generation sequencing. Endogenous retroviruses (ERVs) as a member of transposable elements have been involved in functional roles of various genes in variety organisms. In this study, we identified an Olive flounder ERV (OF-ERV5) using RepeatMasker program, and examined expression pattern of pol gene of OF-ERV5, which indicated the high expression in two kidney samples (head kidney and body kidney). In addition, the 5′LTR sequences of OF-ERV5 are cloned into pGL4.11 vectors to confirm promoter activity. Luciferase assay indicated that the OF-ERV5_LTR showed promoter activity in both HepG2 and HINAE cell lines. These data could be of great use for further study to understand biological function of transposable elements in Oliver flounder.     

一株牙鲆源黏质沙雷氏菌YP1的分离鉴定及致病性分析

黏质沙雷氏菌(Serratia marcescens)是引起人类、动物及植物感染的重要条件致病菌,但其作为鱼类致病菌却鲜有报道。【目的】本研究以从患病牙鲆(Paralichthys olivaceus)病灶处分离的一株黏质沙雷氏菌YP1为研究对象,分析黏质沙雷氏菌对鱼类的致病性及对疾控的影响。【方法】利用形态学、分子生物学及生理生化实验综合鉴定菌株YP1;利用菌株YP1进行人工感染实验、组织病理实验及药敏试验,研究其感染症状、组织病理学、毒力和药物敏感性。【结果】分离自患病牙鲆体表溃疡病灶处的菌株YP1鉴定为黏质沙雷氏菌。感染实验结果显示,牙鲆和斑马鱼的半数致死量(LD₅₀)分别为3.44×10⁷CFU/g和6.28×10⁵CFU/g,除牙鲆外菌株YP1对其他鱼类也具有高致病性;菌株YP1主要导致牙鲆腹水,同时伴有呼吸急促、摄食减弱、脱肛、白便、鳃缺血及多脏器膨大出血等症状,并随着感染时间的延长对脏器损伤呈加重趋势。病理组织切片结果显示,菌株YP1对牙鲆鳃、肠、肝、脾、肾、心均造成损伤。药敏试验结果表明,YP1对左氧氟沙星、诺氟沙星等14种药物敏感;但对氨苄西林、头孢拉定等19种药物具有耐药性。【结论】本研究结果证实了黏质沙雷氏菌是能导致牙鲆腹水病的一种病原菌,同时对其他鱼类也具高致病性,为该菌感染鱼类导致疾病的检测、鉴别和防治提供科学依据。     

Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

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.     

Molecular analysis of complement component C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus

 The amino acid sequences of the human terminal complement components show extensive structural similarity to each other. In this study the C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, were cloned and analyzed. The derived deduced amino acid sequences of the two terminal components were homologous to those of humans, in that the sequences of both species contained LDL receptor, EGF precursor, and two thrombospondin domains. Japanese flounder C9 was found to have a second thrombospondin region in the C-terminus, similar to that reported for rainbow trout and pufferfish. Moreover, these two complement component cDNAs of Japanese flounder had partial similarity to human perforin. These findings show that Japanese flounder C8β and C9 have similar structures, which supports the hypothesis that the terminal complement genes originated from the same ancestral gene. Collectively, these features emphasize the strong similarity among the members of the terminal complement family. Received: 23 March 1999 / Revised: 1 June 1999     

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 ()     

Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.