Takuma Sugaya

Variable region of betanodavirus RNA2 is sufficient to determine host specificity.

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.

Population genetic structure and variability of Pacific herring Clupea pallasii in the stocking area along the Pacific coast of northern Japan

The genetic diversity of wild and hatchery-released Pacific herring Clupea pallasii collected from three brackish lakes and two bays in Honshu and Hokkaido, Japan was examined with five microsatellite loci. All loci showed high genetic variability with expected heterozygosities ranging from 0.815 to 0.945. Significant differences in genotypic and allelic distributions were detected among all locations except for between the two bays in Honshu Island. Pairwise population analysis based on the FST> values showed close genetic relationships among the locations in Hokkaido Island, and the hierarchical analyses of molecular variance showed significant genetic difference between the two islands. Those results suggest the existence of subpopulations due to natal homing. In addition, stocked fish showed as much genetic diversity as the wild fish. The pairwise population analyses also showed close relationships between the hatchery fish and the wild fish in respective stocking areas, showing that no effects of stocking programs on genetic diversity of wild populations were detected.

Genetic effects of hatchery fish on wild populations in red sea bream Pagrus major (Perciformes, Sparidae) inferred from a partial sequence of mitochondrial DNA

Variation in the mitochondrial DNA transcriptional control region sequence was investigated in wild and hatchery-released red sea bream Pagrus major from Kagoshima Bay, where an extensive hatchery-release programme has been conducted for >30 years. The programme has successfully augmented commercial catches in the bay (released juveniles have been produced from the captive broodstock, repeatedly used over multiple generations). Samples were also obtained from outside the bay, where limited stocking has occurred. Genetic diversity indices measured as number of haplotypes, haplotype richness, haplotype diversity and nucleotide diversity were lower in hatchery-released fish than in wild fish. Genetic differences in wild fish from the bay, especially in the inner bay, compared with fish from outside the bay were detected in terms of decreased genetic diversity indices and changed haplotype frequencies. Unbiased population pair-wise F(ST) estimates based on an empirical Bayesian method, however, revealed low genetic differentiation between samples from the bay and its vicinity. Mixed stock identification analyses estimated the proportion of hatchery-released fish in wild populations in the inner and central bays at 39·0 and 8·7%, respectively, although the precision of the estimates was very low because of the small genetic differentiation between populations and relatively small sample sizes. Hence, the long-term extensive hatchery release programme has affected the genetic diversity of wild populations in the bay; however, the genetic effects were low and appeared to remain within the bay.

Automated screening and primer design of fish microsatellite DNA loci on pyrosequencing data

In recent years, massive sequencing approaches have allowed us to determine genomic structures of various organisms rapidly, raising novel applicability of the high-throughput sequence data obtained to various fields of biological studies. We present here a pipeline to search for microsatellite DNA and design PCR primers encompassing the microsatellites on genomic sequence data produced by 454 pyrosequencing. We tested this pipeline, called ‘Auto-primer’, on several fish genomic sequences and obtained many and various candidates for microsatellite DNA loci useful for detecting intraspecies genetic variability. This in silico search for microsatellite DNA is superior to conventional cloning methods, since any sequence patterns of repeat unit can be screened.

Visualization of Corticotropin-Releasing Factor Neurons by Fluorescent Proteins in the Mouse Brain and Characterization of Labeled Neurons in the Paraventricular Nucleus of the Hypothalamus

Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.

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

Bluefin 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.

Characterization of bacterial community composition in rotifer cultures under unexpected growth suppression

Unexpected growth suppression is common in rotifer mass culturing, and bacteria present in culture tanks are believed to be one of the main causes. We used a polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) gene finger-printing method to determine the bacterial community composition in water and that in association with rotifers present in tanks when rotifer growth was suppressed. Bacterial 16S rRNA genes representing Nautella sp. and Marinomonas sp. were identified as dominant in control tanks with good rotifer growth. In contrast, the bacterial community composition was more diverse at start-up in tanks where rotifer growth was suppressed. The community composition changed during rotifer cultivation, and bacterial sequences representative of Ruegeria sp. and Hyphomonas sp. were dominant when rotifer growth recovered. These findings indicate that the bacterial community composition varied according to the rotifer growth conditions and suggest that the initial microbial flora present in the culture water may cause rotifer growth suppression.

Characterization of 12 polymorphic microsatellite DNA loci in the blue shark, Prionace glauca, isolated by next generation sequencing approach

Genetic markers estimating the genetic diversity and population structure of the blue shark (Prionace glauca, Carcharhinidae) have been limited, although this species is one of the most common fishes caught incidentally in the open ocean. A total of twelve novel microsatellite markers in the blue shark were developed using a next generation sequencing approach. All the microsatellite loci isolated were polymorphic with 2–19 alleles, with the observed and expected heterozygosities of 0.25–0.90 and 0.35–0.92, respectively. Cross-species amplification in three other carcharhinid sharks was successful in ten out of twelve loci. The developed microsatellite markers will be useful to analyze the population genetic structure of the carcharhinid sharks including the blue shark.

Application of massively parallel pyrosequencing to the discovery of non-dinucleotide microsatellites: tetra- and pentanucleotide repeat markers for the Pacific abalone (Haliotis discus hannai)

The accelerated progress of ultra high-throughput DNA sequencing technologies has facilitated the process of identifying microsatellites in the genomes, attenuating laborious experimental procedures involved in traditional approaches. We applied a massively parallel pyrosequencing technique to the development of tetra- and pentanucleotide microsatellite markers for the Pacific abalone Haliotis discus hannai.

Development of 15 polymorphic microsatellite markers for the shortfin mako, Isurus oxyrinchus, and cross-species amplification in lamniforme sharks

The shortfin mako (Isurus oxyrinchus, Lamniformes) is one of common sharks caught incidentally by tuna longline and drift net fisheries in the tropical and temperate waters. However, their genetic diversity and population structure for effective management remain to be elucidated with sufficient number of genetic markers. A total of fifteen novel microsatellite markers for the shortfin mako were developed using a next generation sequencing approach. All the microsatellite loci isolated were polymorphic with 2–23 alleles, with the observed and expected heterozygosities of 0.06–1.00 and 0.06–0.93, respectively. Cross-species amplification in six other lamniforme sharks was successful at thirteen out of fifteen loci isolated. The developed microsatellite markers will be useful for the population genetic analysis of lamniforme sharks including the shortfin mako.