A functional genomics tool for the Pacific bluefin tuna: Development of a 44K oligonucleotide microarray from whole-genome sequencing data for global transcriptome analysis

AbstractBluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis, has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production

Viral nervous necrosis (VNN) as a critical infectious disease of orange-spotted grouper, Epinephelus coioides, in the Philippines

Orange-spotted grouper, Epinephelus coioides, is a valuable commodity in the Philippines. In 2001, mass mortality occurred in the grouper larvae at Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC/AQD) and the disease was identified as viral nervous necrosis (VNN). Since then, the disease has been observed every year and the grouper hatcheries have been devastated. In this paper, recent studies of VNN which were conducted at the SEAFDEC/AQD from 2001 to 2006 are reviewed. 1) Susceptibility to the VNN virus was tested among fish species that were cultured in mangrove brackish are. Five representative cultured fish species including orange-spotted grouper, Asian sea bass (Lates calcarifer), mangrove red snapper (Lutjanus argentimaculatus), milkfish (Chanos chanos) and rabbitfish (Siganus guttatus) were used in the test where the virus was intraperitoneally injected into the juveniles. Although low or no mortality occurred in the challenge test, histopathological changes were observed in the brain and retina where the virus was re-isolated. The results were the same among the species except for rabbitfish which had no evidence for the infection. It was verified that the virus has a wide host range. 2) To estimate the possible risk of viral spread by vertical transmission, virus distribution was determined in asymptomatic groupers including 7 broodstock and 17 juveniles with body weights ranging from 4 to 12 kg and 2 to 9 respectively. The virus was detected by PCR method. The highest detection rate was in the brain, and the virus was also detectable in other organs such as the gills, heart, spleen, kidney, blood, esophagus, stomach, intestine, liver, gonad, swim bladder and/or skin. 3) As a possible VNN vaccine, a DNA p;asmid encoding the capsid protein of the virus was evaluated. After the challenge, the mortalities between the native and DNA-injected fish appeared significantly different (P<0.05).This study was supported by funds from Japan International Research Center for Agricultural Sciences (JIRCAS) Research Project "Studies on Sustainable Production Systems of Aquatic Animals in Mangrove Brackish Areas"

Expression profiles of TCRβ and CD8α mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp

AbstractOur previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8α and TCRβ genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08–1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8α and TCRβ mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8α and TCRβ mRNAs. In addition, mRNA expressions of CD8α and TCRβ in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals

Comprehensive validation of T- and B-cell deficiency in rag1-null zebrafish: Implication for the robust innate defense mechanisms of teleosts

Abstractrag1−/− zebrafish have been employed in immunological research as a useful immunodeficient vertebrate model, but with only fragmentary evidence for the lack of functional adaptive immunity. rag1-null zebrafish exhibit differences from their human and murine counterparts in that they can be maintained without any specific pathogen-free conditions. To define the immunodeficient status of rag1−/− zebrafish, we obtained further functional evidence on T- and B-cell deficiency in the fish at the protein, cellular, and organism levels. Our developed microscale assays provided evidence that rag1−/− fish do not possess serum IgM protein, that they do not achieve specific protection even after vaccination, and that they cannot induce antigen-specific CTL activity. The mortality rate in non-vaccinated fish suggests that rag1−/− fish possess innate protection equivalent to that of rag1+/− fish. Furthermore, poly(I:C)-induced immune responses revealed that the organ that controls anti-viral immunity is shifted from the spleen to the hepatopancreas due to the absence of T- and B-cell function, implying that immune homeostasis may change to an underside mode in rag-null fish. These findings suggest that the teleost relies heavily on innate immunity. Thus, this model could better highlight innate immunity in animals that lack adaptive immunity than mouse models.</jats:p

A Genetic Linkage Map for the Tiger Pufferfish, Takifugu rubripes

The compact genome of the tiger pufferfish, Takifugu rubripes (fugu), has been sequenced to the “draft” level and annotated to identify all the genes. However, the assembly of the draft genome sequence is highly fragmented due to the lack of a genetic or a physical map. To determine the long-range linkage relationship of the sequences, we have constructed the first genetic linkage map for fugu. The maps for the male and female spanning 697.1 and 1213.5 cM, respectively, were arranged into 22 linkage groups by markers heterozygous in both parents. The resulting map consists of 200 microsatellite loci physically linked to genome sequences spanning ∼39 Mb in total. Comparisons of the genome maps of fugu, other teleosts, and mammals suggest that syntenic relationship is more conserved in the teleost lineage than in the mammalian lineage. Map comparisons also show a pufferfish lineage-specific rearrangement of the genome resulting in colocalization of two Hox gene clusters in one linkage group. This map provides a foundation for development of a complete physical map, a basis for comparison of long-range linkage of genes with other vertebrates, and a resource for mapping loci responsible for phenotypic differences among Takifugu species