KOBAYASHI Toru

Researcher Information

Degree

• Doctor(Agriculture)（1997/02 The University of Tokyo, Graduate School for Agricultural and Life Sciences）

URL

https://www.nara.kindai.ac.jp/laboratory/suisanseibutuken-hp/kobayashi.html

J-Global ID

• 200901074432751815

Research Interests

• Amago salmon   Rainbow trout   Gynogenetic Breeding   Genome Editing   Morphogenesis   Early development   chimera   Primordial germ cell   Cell transplanting   Willow minnow   Clone   Sex differentiation   Reproduction   Breeding   Polｙploidy   染色体操作   Chromosome manipulation   

Research Areas

• Life sciences / Aquaculture / Fish reproductive physiology
• Life sciences / Aquaculture / Fish Breeding and Genetics
• Life sciences / Developmental biology
• Life sciences / Cell biology
• Life sciences / Molecular biology

Academic & Professional Experience

• 2012/04 - Today  Lab. for Aquatic Biology, Faculty of Agriculture, Kinki UniversityProfessor
• 2007/04 - 2012/03  Kindai UniversityFaculty of Agriculture
• 2004/04 - 2007/03  Lab. for Aqauctic Biology, Faculty of Agriculture, Kinki UniversityAsistant professor
• 2000/04 - 2004/03  Lab. for Aqauctic Biology, Faculty of Agriculture, Kinki UniversityLecturer
• 1997/04 - 2000/03  Lab. for Aqauctic Biology, Faculty of Agriculture, Kinki UniversityReearch asociate
• 1993/04 - 1997/03  Shiga prefecture fisheries experimental station増殖係senior technical staff
• 1990/04 - 1993/03  Shiga prefecture Samegai Trout Farmsenior technical staff
• 1985/04 - 1990/03  Shiga prefecture, Samegai Trout FarmTechnical staff

Education

•        - 1985/03  Kindai University  Faculty of Agriculture  Department of Fisheries

Association Memberships

• The Japanese Society of Germone Editing   the Zoological Society of Japan   the Japanese Society of Animal Breeding and Genetics   Japanese Society for Marine Biotechnology   Japanese Society for Aquaculture Science   The Ichthyoligical Society of Japan   The Japanese Society of Fisheries Science   

Published Papers

• Efficient Production of Doubled Haploids as Isogenic Line Founders via Double Heat Shock Treatment in Willow Gudgeon (Gnathopogon caerulescens)

  Toru Kobayashi; Akito Takeda; Kanta Hara

  Aquaculture Research Hindawi Limited 2023 1 - 17 1355-557X 2023/02 [Refereed]
   

  Doubled haploids (DHs) are essential founders for breeding via isogenic line production. We determined the optimal timing of the first and second (HST1 and HST2) double heat shock treatment for DH production in the willow gudgeon (Gnathopogon caerulescens). Gynogenesis was induced by fertilisation with UV-irradiated sperm (20 mJ/cm2). Each HST was performed at 40.5°C for 1 min. In experiment I, HST1 was applied 23–31 min after the induction of gynogenetic development (incubated at 20°C). HST2 was performed 15 min after HST1 treatment. When HST1 was started 25–26 min after activation, normally hatched larvae appeared relatively frequently (35.6–100%), and most were DHs. In experiment II, HST1 was started 26 min after activation, and HST2 was started 10–25 min after HST1. Starting HST2 10 min after HST1 resulted in 33% more normally hatched larvae than starting 22.5–25 min after. DHs were prevalent among hatched larvae (45.1% and 20.8%, respectively) when performing HST2 10–12.5 min after HST1. Under appropriate HST1 and HST2 timing, gynogens were at the zygote stage, early prophase, and prophase or early prometaphase of the first cell cycle. These results establish the appropriate timing of HST1 and HST2 for the induction of willow gudgeon DHs.
• Targeted deletion of ecto-5′-nucleotidase results in retention of inosine monophosphate content in postmortem muscle of medaka (Oryzias latipes)

  Yu Murakami; Masashi Ando; Ryota Futamata; Tomohisa Horibe; Kazumitsu Ueda; Masato Kinoshita; Toru Kobayashi

  Scientific Reports Springer Science and Business Media LLC 12 (1) 18588  2022/11 [Refereed]
   

  Abstract Inosine monophosphate (IMP) is an important indicator of meat freshness and contributes to its umami taste. An attractive strategy for enhancing umami is to suppress the IMP-degrading activity and increase the IMP content in the skeletal muscle through genome editing technology using the CRISPR-Cas9 system. However, the molecular mechanisms underlying IMP degradation remain unclear. We cloned two ecto-5′-nucleotidase genes, designated as ecto-5′-nucleotidase-a (nt5ea) and ecto-5′-nucleotidase-b (nt5eb), from medaka (Oryzias latipes), a vertebrate model organism. Expression analysis using embryos showed that nt5ea or nt5eb overexpression remarkably upregulated IMP degradation, and that the IMP-degrading activity was higher in Nt5ea than in Nt5eb. Furthermore, we established frame-shifted or large deletion (lacking nt5ea or nt5eb locus) mutant strains and assayed the effects of gene disruptions on the amount of IMP in skeletal muscle. The nt5ea-deficient medaka showed considerable higher levels of IMP at 48 h postmortem than did the wild-type fish. The nt5eb mutants also exhibited higher IMP contents than that in the wild types, but the increase was less than that in the nt5ea mutants. Our results demonstrated that nt5e is an important regulator of IMP levels in skeletal muscle and that its loss of function was effective in maintaining IMP content.
• An effective double gene knock‐in strategy using small‐molecule L755507 in the medaka fish ( Oryzias latipes)

  Yu Murakami; Toru Kobayashi

  genesis Wiley 1526-954X 2022/01 [Refereed]
  
• Gonad formation and pre-meiotic proliferation of gonocytes in the Japanese willow minnow Gnathopogon caerulescens

  KOBAYASHI Toru; ASHIDA Hiroshi

  Aquaculture Science 67 (1) 9 - 17 2019/04 [Refereed]
  
• Double heat shock more potently suppresses cleavage of fertilized willow minnow (Gnathopogon caerulescens) eggs than single heat shock treatment.

  KOBAYASHI Toru; FUJII Hikaru

  Aquaculture Research 2019 [Refereed]
  
• Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares

  Toru Kobayashi; Tomoki Honryo; Yasuo Agawa; Yoshifumi Sawada; Ileana Tapia; Karla A. Macias; Amado Cano; Vernon P. Scholey; Daniel Margulies; Naoki Yagishita

  REPRODUCTIVE BIOLOGY INST ANIMAL REPRODUCTION FOOD RESEARCH 15 (2) 106 - 112 1642-431X 2015/06 [Refereed]
   

  To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 +/- 3.2 cells), compared to that at 1 dph (11 +/- 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species. (C) 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Sp. z o.o. All rights reserved.
• Identification of male sex-linked DNA sequence of the cultured Pacific bluefin tuna Thunnus orientalis

  Yasuo Agawa; Mayui Iwaki; Takafumi Komiya; Tomoki Honryo; Kouhei Tamura; Tsukasa Okada; Naoki Yagishita; Toru Kobayashi; Yoshifumi Sawada

  FISHERIES SCIENCE SPRINGER JAPAN KK 81 (1) 113 - 121 0919-9268 2015/01 [Refereed]
   

  A male-specific marker of a DNA sequence for the Pacific bluefin tuna Thunnus orientalis, Male delta 6 (Md6), was identified by comparative study of the sequences obtained by F3-cultured male and female AFLP-selective DNA amplification products, followed by high-throughput DNA sequencing. Md6 was characteristic in continuous 6-bp nucleotide deletions compared to redundant sequences that could be seen in both sexes. The PCR genotyping primers were developed based on the Md6 sequence, which showed that 29 of 32 F3 males (90.6 %) and 9 of 13 (69.2 %) F2 parental males were genotyping positive, whereas in females, the genotyping test was negative in parental F2 (n = 12), and 1 of 32 (3.1 %) F3 female was Md6-positive. Parentage allocation tests suggested that Md6-positive F2 males were more attendant in spawning than Md6-negative males, suggesting that Md6 was passed from male parents to male progeny under aquaculture conditions.
• Genetic population structure of the Pacific bluefin tuna Thunnus orientalis and the yellowfin tuna Thunnus albacares in the North Pacific Ocean

  Shohei Nomura; Toru Kobayashi; Yasuo Agawa; Daniel Margulies; Vernon Scholey; Yoshifumi Sawada; Naoki Yagishita

  FISHERIES SCIENCE SPRINGER JAPAN KK 80 (6) 1193 - 1204 0919-9268 2014/11 [Refereed]
   

  The genetic population structure of the Pacific bluefin tuna (PBF) Thunnus orientalis and the yellowfin tuna (YFT) T. albacares in the North Pacific Ocean was investigated. The polymorphism of microsatellite (SSR) loci and sequences of mitochondrial DNA control region (mtCR) were analyzed for 71 samples of PBF from Japan and Mexico and 45 samples of YFT from Japan and Panama. In the SSR analyses, both single-locus (-0.010 to 0.008 in PBF and -0.023 to 0.020 in YFT) and global multilocus (0.003 in PBF and -0.002 in YFT) F-ST values among the geographic populations were low and not significant in these species. In the mtCR analyses, neither the neighbor-joining tree nor the minimum spanning network showed genetic differentiation among the geographic populations in each species. The pairwise F-ST values among the geographic populations of them (-0.005 in PBF and -0.020 to -0.014 in YFT) were low and not significant. Our SSR and mtCR data suggested that genetic differentiations were not evident among the eastern and western populations in the North Pacific Ocean either in PBF or in YFT. Mismatch distributions, demographic parameters, and neutrality tests suggested that sudden population expansion of PBF and YFT in the North Pacific Ocean occurred 628,000-731,000 and 450,000-525,000 years ago, respectively.
• Effect of Water Temperature on Embryonic Development of Yellowfin Tuna Thunnus albacares Inhabiting the Eastern Pacific Ocean

  GUILLEN ANGEL; HONRYO TOMOKI; IBARRA JUAN; CANO AMADO; MARGULIES DANIEL; SCHOLEY VERNON P; WEXLER JEANNE B; STEIN MARIA S; KOBAYASHI TORU; SAWADA YOSHIFUMI

  Aquaculture Science 62 (3) 319 - 322 0371-4217 2014/09 [Refereed]
  
• Isolation and characterization of 25 microsatellite loci from the Pacific bluefin tuna Thunnus orientalis (Perciformes, Scombridae)

  Naoki Yagishita; Yoshifumi Sawada; Yasuo Agawa; Toru Kobayashi

  CONSERVATION GENETICS RESOURCES SPRINGER 6 (1) 189 - 191 1877-7252 2014/03 [Refereed]
   

  The pacific bluefin tuna (PBT) Thunnus orientalis is one of the most important species for fisheries in the world. This species has been highly exploited in fisheries, resulting in dwindling of its natural resources. The artificial hatching of a third generation of fully cultured PBT was a success in 2007, suggesting the possibility of mass production of PBT for food and for seed release. To enhance stock structure studies and investigate genetic feature of seedlings, we isolated 25 polymorphic microsatellite loci (2-18 alleles/locus; expected heterozygosity, 0.042-0.941) from PBT.
• Molecular identification and expression of FOXL2 and DMRT1 genes from willow minnow Gnathopogon caerulescens

  Hiroshi Ashida; Naoki Ueyama; Masato Kinoshita; Toru Kobayashi

  REPRODUCTIVE BIOLOGY INST ANIMAL REPRODUCTION FOOD RESEARCH 13 (4) 317 - 324 1642-431X 2013/12 [Refereed]
   

  Willow minnow, Gnathopogon caerulescens, is a commercially important freshwater fish species in Japan. In particular, female fish with eggs are more expensive than male fish. Many methods have been tested to achieve 100% willow minnow female production; however, none of them were successful because of the temperature-dependent sex determination mechanism. To date, the molecular mechanism underlying sex determination in willow minnow is still not completely understood. This study was designed as an attempt to understand the sex differentiation mechanism of willow minnow at the molecular level. We cloned the mRNAs of two sex-related genes of G. caerulescens forkhead-box protein L2 (FOXL2) and doublesex- and mab-3-related transcription factor 1 (DMRT1). We compared the inferred amino acid sequences to those of other fish and examined the expression patterns of these genes in adult tissues and during development. The FOXL2 and DMRT1 genes of willow minnow were isolated and identified. Both gene sequences share high homology with those of Chinese Cyprinidae. The cell- and tissue-specific expression of FOXL2 and DMRT1 were also determined. Both genes were expressed in gonads, and their expression level increased before gonadal sex differentiation. Overall, these results suggest that these two genes play a key role in the sex differentiation of willow minnow. Detailed knowledge regarding this mechanism will be useful for efficient all-female cyprinid fish production that follows a temperature-dependent sex determination system. (C) 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
• 〈Original〉Sampling record of aquatic animals from Sakatahana, Shirahama-cho, Wakayama Prefecture, Japan

  宮口修平; 田島灯台; 小林 徹; 柳下直己

  近大農紀要 近畿大学農学部 46 (46) 37 - 53 0453-8889 2013/03 

  [Synopsis] The members of the Laboratory for Aquatic Biology, Faculty of Agriculture, Kinki University collected aquatic animals at Sakatahana, Shirahama-cho, Wakayama Pref. during field practice every summer from 2007 to 2011. Here, we made a provisional list of the collected specimens of fishes, crustaceans, gastropods, and bivalves that include 58, 37, 27, and 12 species, respectively. Among them, the numbers of the species of temperate to subtropical waters were 36 in fishes, 13 in gastropods, and 4 in bivalves.
• Novel method for improving the antioxidative properties of fish meat by direct injection of sodium l-ascorbate into the blood vessels of live fish

  Yasuyuki Tsukamasa; Keisuke Kato; Bimol Chandra Roy; Yasunori Ishibashi; Toru Kobayashi; Tomohiro Itoh; Masashi Ando

  Fisheries Science 79 (2) 349 - 355 0919-9268 2013 [Refereed]
   

  The color of the red muscle of pelagic fish is used as an indicator of freshness, and sustaining a bright red color is important for maintaining the commercial value of pelagic fish. Feeding fish with antioxidant compounds can delay metmyoglobin (metMb) formation, but the process requires long-term feeding. Live cultured Japanese horse mackerel Trachurus japonicus were used in this study. After anesthetization, 2 ml of blood was drawn from blood vessels, and a parenteral solution including an antioxidant compound was injected. Browning, metMb formation, and lipid oxidation of dark muscle during chilled storage were delayed by injecting sodium l-ascorbate as an antioxidant compound in the blood vessels of live fish. © 2013 The Japanese Society of Fisheries Science.
• Molecular identification and tissue distribution of peroxisome proliferators activated receptor gamma transcript in cultured Thunnus orientalis

  Yasuo Agawa; Tomoki Honryo; Ayako Ishii; Toru Kobayashi; Hiromi Oku; Yoshifumi Sawada

  AQUACULTURE RESEARCH WILEY-BLACKWELL 43 (8) 1145 - 1158 1355-557X 2012/07 [Refereed]
   

  Pacific bluefin tuna (PBT) peroxisome proliferator-activated receptor ? (PPAR?) gene was characterized to know the expression of its transcript contribution to the development of PBT, because PPAR? is the key molecule for adipose cell differentiation. Resulting cDNA and deduced amino acid sequence had high similarities to other teleosts that consisted of A to F domains. Phylogenetic tree analysis indicated a close relationship PBT PPAR? to cobia and flatfishes ones among the teleosts with the similarity of characteristic insertion sequences at amino terminal region of E/F domains. PPAR? transcripts quantification profile in the tissues of a 13-month-old PBT indicated the correlation between its quantity with the muscle types of different lipid content. The transcripts were also detected in the head kidney, digestive organs and red muscle with higher level inferred the PPAR? contribution to the multiple physiological processes. PPAR? transcripts were quantified for PBT juveniles fed artificial diets to investigate the effect of phospholipid supplementation on the growth, survival and PPAR? expression, to verify the efficacy of phospholipid source. No obvious difference in growth performance, survival and gene expression of PBT juveniles was identified, this may be due to DHA/EPA ratio analogy between diets and indicating that phospholipid sources in the juvenile diet might be adaptable.
• Seasonal distribution of adult crucian carp nigorobuna Carassius auratus grandoculis and gengoroubuna Carassius cuvieri in Lake Biwa, Japan

  Yoshio Kunimune; Yasushi Mitsunaga; Kazuyoshi Komeyama; Masanari Matsuda; Toru Kobayashi; Tsutomu Takagi; Takeshi Yamane

  FISHERIES SCIENCE SPRINGER TOKYO 77 (4) 521 - 532 0919-9268 2011/07 [Refereed]
   

  Seasonal habitat use by nigorobuna Carassius auratus grandoculis Temminck et Schlegel and gengoroubuna Carassius cuvieri (Temminck et Schlegel) in Lake Biwa was investigated using acoustic telemetry. Twenty-three nigorobuna and 11 gengoroubuna specimens caught using set-nets in the lake's south basin were surgically fitted with acoustic transmitters and then released. Signals from the fishes were recorded by 23 receivers installed around the lake. Between April and June 2007, the first spawning season after release, signals were received from all tagged fishes. Thereafter, until the second spawning season, signals were collected from 26 and 45% of released nigorobuna and gengoroubuna individuals, respectively. Seasonal habitat preferences for these species were studied by distance-based analysis. The analysis revealed that nigorobuna tended to stay near their spawning area in the south basin of the lake throughout the year, whilst gengoroubuna tended to show a seasonal migration pattern between the north and south basins. After the spawning season, the latter species migrated to the north basin where it remained until the next spawning season, when it returned to the south basin. This is the first report of seasonal migration of nigorobuna and gengoroubuna in Lake Biwa.
• Gonadal morphogenesis and sex differentiation in cultured chub mackerel, Scomber japonicus

  Toru Kobayashi; Ryo Ishibashi; Shinji Yamamoto; Satoshi Otani; Koichi Ueno; Osamu Murata

  AQUACULTURE RESEARCH WILEY-BLACKWELL PUBLISHING, INC 42 (2) 230 - 239 1355-557X 2011/01 [Refereed]
   

  The timing of primordial germ-cell (PGC) migration with regard to the gonadal anlagen, gonad formation and sex differentiation was examined histologically in the chub mackerel (Scomber japonicus) at 5-190 days post hatching (dph). At 5 dph, PGCs appeared on the peritoneal epithelium surface or in the mesentery, on the dorsal side of the abdominal cavity. By 10 dph, stromal cells around the PGCs proliferated. The gonadal primordium was formed by 15 dph. The gonadosomatic index was 0.01% at 30 dph and increased thereafter (0.32% in females and 0.04% in males at 160 dph). Ovarian differentiation occurred at 30-40 dph, indicated by ovarian cavity formation (elongation and fusion of the upper and lower ovarian edges). Meiosis was subsequently initiated. A few meiotic oocytes surrounded the cavity at 50 dph; most were in the perinucleolus stage at 60 dph and attained a diameter of 60-70 mu m at 190 dph. Testicular differentiation occurred at 30 dph, indicated by the formation of the sperm duct primordium. Spermatogonia gradually proliferated, developing into spermatocytes at the chromatin-nucleolus stage (after 90 dph) and subsequently into spermatids and spermatozoa (160 dph). These data could aid the development of seeding and cell-engineering technologies for scombrid fish.
• Production of Gynogenetic Inbred Lines of Rainbow Trout (Oncorhynchus mykiss) and Selection for Early Spawning Time and Large Egg Size

  KOBAYASHI TORU; FUSHIKI SHOZO

  Aquaculture Science 水産増殖談話会 59 (2) 191 - 198 0371-4217 2011 [Refereed]
   

  醒井養鱒場ニジマス血統集団から選抜された個体を用いた雌性発生継代によって、早期産卵形質および大型卵産出形質に関する遺伝的固定をおこなった。夜間照明による成熟調節操作を受けた1986年夏季産卵群（P）の再生産形質のうち排卵時期と産出卵のサイズの分布を調べ、産卵期間のうちで早期、中期、晩期に排卵した個体の中から大型卵を産出した個体をそれぞれ選抜し、それらの卵を用いて雌性発生で継代した。雌性発生1代目（1G）は1988年の秋に成熟し、それぞれの次世代集団は各選抜親魚の産卵期および大型卵産出に関する形質をよく受け継いでいた。そのうち特に早期に大型卵を産出したC系統の個体を用いて雌性発生継代した2代目（2G）は醒井通常系統よりも6週間も早い9月20日から排卵を開始した。1Gの卵サイズは系統ごとに広くばらついた。雌性発生C系統の1代目の平均卵径は4.90mmとP群の1.06倍であり、その実現遺伝率は0.164であった。
• Cytological mechanisms of induced gynogenesis in nigorobuna crucian carp eggs exposed to heat or cold shock for retention of second polar body.

  Kobayashi T; Nemoto M

  Aquaculture Science 水産増殖談話会 58 (3) 337 - 343 0371-4217 2010 [Refereed]
   

  ニゴロブナにおける雌性発生卵の第二極体放出阻止のための低温刺激処理（CST）および高温刺激処理（HST）後の染色体の行動を観察した。受精7日後の正常仔魚生存率はCSTおよびHSTをそれぞれ媒精後7分および9分に開始した場合に最大値を示した。雌性発生は1腹卵を使って紫外線照射精子（3,000erg／mm2）で誘導した。CSTの後、第二減数分裂の紡錘体が消失し、カリオメアと半数性核、あるいは二倍性核が、雌性前核と極体の代わりに観察された。これらの構造体は処理後15分（媒精後62分）に精子星状体の中心へ移動して接合核を形成し、媒精後72分に第1卵割が起こった。一方HST後では、卵割は処理後12分に起こった。核の変化はCST後よりもHST後の方が早かったが、両処理後の核の行動は類似していた。カリオメアおよび前核の行動の違いは処理の違いではなく、卵の熟度と感受性の違いによっていると考えられた。
• Molecular cloning and daily mRNA levels of prolactin and somatolactin in aquacultured Pacific bluefin tuna (Thunnus orientalis)

  Kohsuke Adachi; Keitaro Kato; Masayoshi Yamamoto; Katsuya Ishimaru; Toru Kobayashi; Osamu Murata; Hidemi Kumai

  AQUACULTURE ELSEVIER SCIENCE BV 296 (1-2) 110 - 116 0044-8486 2009/11 [Refereed]
   

  We cloned the prolactin (PRL) and somatolactin (SL) genes of Pacific bluefin tuna (PBT: Thunnus orientalis) and investigated their daily mRNA expression patterns under aquacultured conditions. The PRL and SL cDNAs of PBT encode 211 and 231 aa prehormones which give rise to 187 and 207 aa mature peptides, respectively, and which belong to the same clades as those of other Perciformes. The highest gene expression levels of PRL and SL in PBT were found at 6 am under natural light conditions, which is about 3 times higher than the constitutive levels in both cases. Interestingly, scatter diagrams show a strictly correlated expression between PRL and SL (r=0.82: p<0.01) while no correlation was found between PRL or SL and the growth hormone (GH). These results suggest that PRL and SL play synchronized role(s) in daily physiological phases of PBT under aquacultured conditions. (C) 2009 Elsevier B.V. All rights reserved.
• Physiological response of triploid rainbow trout to exhaustive exercise.

  T. Kobayashi; Shozo Fushiki; Koichi Ueno

  Aquaculture Science 57 (3) 361 - 370 0371-4217 2009 [Refereed]
  
• Pulsed expression of growth hormone mRNA in the pituitary of juvenile Pacific bluefin tuna under aquacultured conditions

  Kohsuke Adachi; Keitaro Kato; Masayoshi Yamamoto; Katsuya Ishimaru; Toru Kobayashi; Osamu Murata; Hidemi Kumai

  AQUACULTURE ELSEVIER SCIENCE BV 281 (1-4) 158 - 161 0044-8486 2008/09 [Refereed]
   

  The daily expression profile of growth hormone (GH) mRNA in juvenile Pacific bluefin tuna (Thunnus orientalis) was investigated under aquacultured conditions. Total RNA from pituitaries (n=5) was sampled from 10 am to 12 pm the next day at 1 h intervals. The expression levels of GH mRNA were evaluated using real-time PCR normalized against the beta-actin gene. The expression level of GH transcripts reached a peak at 3-4 am at which time it was about 10 times higher than at any other time period. Considering their high growth rate compared to red seabream (Pagrus major) and other aquacultured species which shows continuous CH mRNA expression patterns, it can be concluded that the pulsed expression of GH mRNA just before daybreak is a key for the extremely high somatic growth rate of Pacific bluefin tuna. (C) 2008 Elsevier B.V. All rights reserved.
• Genetic identification of larvae and juveniles reveals the difference in the spawning site among Cyprininae fish species/subspecies in Lake Biwa

  Takashi Suzuki; Toru Kobayashi; Koichi Ueno

  ENVIRONMENTAL BIOLOGY OF FISHES SPRINGER 82 (4) 353 - 364 0378-1909 2008/08 [Refereed]
   

  To understand the differences in the spawning sites among Cyprininae fishes in Lake Biwa, we conducted periodic sampling of larvae and juveniles at three sites (irrigation ditch, St. 1; river, St. 2; and satellite lake, St. 3). On the basis of species/subspecies identification by using RAPD analysis, we examined the species composition of the larvae and juveniles at these three sampling sites. The number of specimens was 616, 68, and 117 at St. 1, St. 2, and St. 3, respectively. Based on morphological and genetic identification, the specimens were found to include nine fish species/subspecies, namely, Carassius auratus grandoculis, Carassius cuvieri, Carassius auratus langsdorfii, Cyprinus carpio, Sarcocheilichthys sp., Silurus asotus, Oryzias latipes, Odontobutis obscura obscura, and Rhinogobius sp. The species composition at the three sites also differed. Among the Cyprininae fishes, C. auratus grandoculis, C. auratus langsdorfii, and Cyprinus carpio were found in abundance at St. 1; C. cuvieri was not collected from St. 1 but was found at the other two sites, particularly St. 3. Among the other fishes, Rhinogobius sp. was collected at St. 1 and St. 3, whereas the other four occurred only at St. 1. These results suggest that the selection of spawning sites by C. cuvieri differs to a certain extent from that of the other Cyprininae fishes, and the irrigation ditch in the lake is an important habitat for the larvae and juveniles of native fish species.
• Isolation and characterization of nine microsatellite loci from the chub mackerel, Scomber japonicus (Perciformes, Scombridae)

  N. Yagishita; T. Kobayashi

  MOLECULAR ECOLOGY RESOURCES BLACKWELL PUBLISHING 8 (2) 302 - 304 1471-8278 2008/03 [Refereed]
   

  The stock abundance of the chub mackerel Scomber japonicus - a very important species for fisheries, particularly in Japan - in the Pacific Ocean off Japan has remained at a low level. For studying the population genetics of the chub mackerel, we isolated nine polymorphic microsatellite loci (12-31 alleles/locus; expected heterozygosity, 0.762-0.983) from this species. Cross-species amplification indicated that eight of the nine microsatellite loci in the blue mackerel S. australasicus were polymorphic and functional.
• A method for the microinjection into naturally spawned eggs of marine fish, especially cultured Pacific bluefin tuna Thunnus orientalis

  Satoshi Otani; Manabu Ohara; Shigeru Miyashita; Toru Kobayashi

  FISHERIES SCIENCE BLACKWELL PUBLISHING 74 (1) 208 - 210 0919-9268 2008/02 [Refereed]
  
• Effect of HCG Injections on One-year-old Captive Chob Mackerel Scomber japonicus

  Ishibashi R; Yamamoto S; Kobayashi T; Izumi K; Miyashita S; Murata O

  Aquaculture Science Japanese Society for Aquaculture Science 55 (1) 17 - 22 0371-4217 2007 [Refereed]
   

  Human chorionic gonadotropin (HCG) was injected to one-year old captive chub mackerel (Scomber japonicus) in June 2005, a timing equivalent to the fish spawning period, to see if fertilized eggs would be formed. The sample fish were accommodated in a net cage and measured for total length, fork length, body length, head length, body depth, body width and body weight every 10 days until 100 days after hatching, then every 30-day thereafter. The sample fish were also checked for ovulation and spermiation by abdominal thrust 280 days after hatching. Their gonadal gland was then removed, weighed and measured for gonad somatic index (GSI) and, for females, KG value. Although spermiation was not confirmed in any males 280 days in age, spermiation was confirmed in half the male fish after 310 days in age and in all the male fish after 340 days in age. For the female fish, no ovulation was observed in individuals 310 days in age, but ovulation was confirmed in 8 out of 10 fish 340 days in age. Five groups of 10 male and 10 female in each received single injection of HCG (200 IU/kg BW) from 360 days in age (1st group) to 376 days in age (5th group) . The fish in four group spawned by the 3 days after the injection. In the group of eggs injected on June 27 (4th group), fertilized eggs were seen, although in a small quantity. When these fertilized eggs were cultured, they were observed to hatch normally and the hatchlings grew successfully.
• Morphological characteristics of nigorobuna Carassius auratus grandoculis called Io in Lake Nishino

  T Suzuki; H Nagano; T Kobayashi; K Ueno

  FISHERIES SCIENCE JAPANESE SOC FISHERIES SCIENCE 71 (3) 679 - 681 0919-9268 2005/06 [Refereed]
  
• Seasonal changes in the number of larvae and juveniles of crucian carps in the reed zone of Lake Biwa based on (sub) species identification using RAPD markers

  T Suzuki; H Nagano; T Kobayashi; K Ueno

  NIPPON SUISAN GAKKAISHI JAPANESE SOC FISHERIES SCIENCE 71 (1) 10 - 15 0021-5392 2005/01 [Refereed]
   

  We investigated species composition, distribution and seasonal changes in the number of larvae and juveniles of crucian carps in the reed zone of Lake Biwa using RAPD markers. A total of 973 larvae and juveniles were collected from the reed zone in a total of nine samplings conducted from April to July, 2002. In the first sampling, 868 larvae and juveniles, mainly preflexion larvae, were collected, and it was found that Gengoroubuna and Ginbuna are distributed within the reed zone. However, Nigorobuna distributed between the center and close to the shore within the reed zone. After the first sampling, larvae and juveniles, mainly postflexion larvae, were collected close to the shore in the reed zone, and most of those were Nigorobuna.
• Improvement of sperm motility of sex-reversed male rainbow trout, Oncorhynchus mykiss, by incubation in high-pH artificial seminal plasma

  T Kobayashi; S Fushiki; K Ueno

  ENVIRONMENTAL BIOLOGY OF FISHES SPRINGER 69 (1-4) 419 - 425 0378-1909 2004/03 [Refereed]
   

  Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60-90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4degreesC, the percentage of motile spermatozoa increased from 0-5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.
• Production of germline chimera in loach (Misgurnus anguillicaudatus) and proposal of new method for preservation of endangered fish species

  M Nakagawa; T Kobayashi; K Ueno

  JOURNAL OF EXPERIMENTAL ZOOLOGY WILEY-LISS 293 (6) 624 - 631 0022-104X 2002/11 [Refereed]
   

  As part of the technique for preservation of endangered fish species, germline chimeras using the loach (Misgurnus anguillicaudatus) model were generated by embryonic cell manipulation. Transplanting cells from early-stage embryos of a wild-type strain to the blastoderm of an orange-type strain generated chimeras. Within three days, many of the resulting individuals exhibited a dark pigment on several parts of their body, characteristic of the donor but not of the orange strain. At one year of age, four chimeras (one female and three males) were pair-mated with the orange type, and F, was obtained. One of the four F-0 chimeras produced progeny similar to the wild type at a frequency of 0.24% (2/841). The results suggest that this method is a promising technique for the preservation of endangered fish species. (C) 2002 Wiley-Liss, Inc.
• Heteromorphic sex chromosomes of lizardfish (Synodontidae): focus on the ZZ-ZW₁W₂ system in Trachinocephalus myops

  K Ueno; K Ota; T Kobayashi

  GENETICA KLUWER ACADEMIC PUBL 111 (1-3) 133 - 142 0016-6707 2001 [Refereed]
   

  The karyotype and DNA content of four lizardfish species (family Synodontidae), that is, Saurida elongata, Synodus ulae, Synodus hoshinonis and Trachinocephalus myops, were analyzed. The karyotype of T. myops significantly differed from that of the other three species having diploid chromosome number of 48 with mainly acrocentric chromosomes and the ZZ-ZW sex chromosome system. The chromosome number of male T. myops was 2n = 26, while that of female T. myops was 2n = 27. The karyotype consisted of 11 pairs of metacentrics, one pair of acrocentrics and, in addition, two large metacentrics in the male and a single large metacentric, a distinctly small subtelocentric and a microchromosome in the female. C-banding demonstrated that in the female the subtelocentric chromosome and the microchromosome were heterochromatic. The karyotype of T. myops was thought to be derived from a 48 chromosome type synodontid fish through the involvement of Robertsonian rearrangement; the rearrangement of the sex chromosomes proceeded during karyotype evolution. Among the chromosomes, the large metacentrics were determined to be neo-Z (a fusion of the original Z and an autosome), the microchromosomes the W-1 (originally W), and the subtelocentric chromosomes the W-2 (derived from an autosome pair). The miniaturization of W-1 and W-2 chromosomes and their heterochromatinization suggested that sex chromosomes in this species have been already highly differentiated. The findings on DNA content implied that the karyotype of T. myops evolved by centric fusion events without loss in DNA amount.
• Effect on Crowding of Water Fleas, MIJINKO, with verious wavelengthlights.

  KOBAYASHI Toru

  Reserch Report of Shiga Prefecture Fisheries Experimental Station 48 27 - 31 2001 [Refereed][Invited]
  
  ダウンロード
• Evolution of heteromorphic sex chromosomes in the order Aulopiformes

  K Ota; T Kobayashi; K Ueno; T Gojobori

  GENE ELSEVIER SCIENCE BV 259 (1-2) 25 - 30 0378-1119 2000/12 [Refereed]
   

  The fish order Aulopiformes contains both synchronously hermaphroditic and gonochoristic species. From the cytogenetic viewpoint, few reports show that gonochoristic Aulopiformes have heteromorphic sex chromosomes. Because fish in this order give us a unique opportunity to elucidate the evolution of sex chromosomes, it is important to examine a phylogenetic relationship in Aulopiformes by both molecular evolutionary and cytogenetic methods. Thus, we conducted molecular phylogenetic and cytogenetic studies of six Aulopiform species. Our results suggested that hermaphroditic species were evolutionarily derived from gonochoristic species. It follows that the hermaphroditic species might have lost the heteromorphic sex chromosomes during evolution. Here, we suggest a possibility that heteromorphic sex chromosomes can disappear from the genome. even if they have appealed once in evolution. Taking into account Ohno's hypothesis that heteromorphic sex chromosomes might have emerged from autosomes. we propose the hypothesis that heteromorphic sex chromosomes may have undergone repeated events of appearance and disappearance during the course of fish evolution. (C) 2000 Elsevier Science B.V. All rights reserved.
• Cytological inspection on the optimum timing for retention of the second polar body in early gynogenetic eggs of amago salmon

  T Kobayashi; K Ueno

  NIPPON SUISAN GAKKAISHI JAPANESE SOC FISHERIES SCIENCE 66 (5) 840 - 845 0021-5392 2000/09 [Refereed]
   

  Behavior of spindle and chromosomes in early development of gynogenetic amago salmon eggs was examined for explication of basic cytological mechanisms to induce gynogenetic diploid using genetically inactivated rainbow trout sperm by W-irradiation and with heat shock treatment (0 degreesC for 20 min) for retention of second polar body. The heat shock treatment that started 30 minutes after insemination of amago salmon eggs incubated at 12.0 degreesC was the optimum condition to induce meiotic gynogenetic diploids compared with other conditions of treatment. At 30 minutes, the eggs were in late anaphase of meiosis II. The percentage of eggs that became diploid larvae when treatment started at 5 to 30 minutes after insemination varied little. When treatment started at anaphase, the survival rate was greater than when treatment started at metaphase. It seems that physical damage to the eggs caused by heat is less at anaphase. The second polar body was retained in fewer eggs when heat treatment started later, in the range of 35 to 60 minutes after insemination. Cytokinesis may have prevented such retention more than the increased distance between chromosomes.
• Cytological observation on behavior of the nuclei after chromosome manipulation during early development of rainbow trout

  T Kobayashi

  NIPPON SUISAN GAKKAISHI JAPAN SOC SCI FISHERIES SCI 64 (5) 782 - 791 0021-5392 1998/09 [Refereed]
   

  Heat shock treatment for 20 min from 15 min after insemination suppressed the formation of the second polar body of the normally developed eggs, and the genetic material of the second polar body was retained in the egg. These processes did not result in the formation of a diploidy female pronucleus, but formed some karyomeres. They were tugged with the female pronucleus toward the male pronucleus already present in the center of the sperm aster. Thus, they adhered, and formed a fertilized nucleic group like a cluster of grapes. The genetic inactivated sperm irradiated with ultraviolet rays (UV-sperm) became swollen and formed a male pronucleus after passing through the micropile. It behaved like normal sperm until the connection with the female pronucleus. The paternal genetic material in gynogenetic embryos disappeared during the anaphase of the first cleavage, caused by the incomplete chromosome formation from the male pronucleus, and the paternal DNA remained near the cleavage plane. In the gynogenetic diploid embryo inseminated with UV-sperm, and polyploidized with heat shock treatment, the above mentioned cytological events caused by dual artificial manipulation occurred in the same egg, i.e., the genetic material formed a triploidy nucleus like a cluster of grapes, and a male pronucleus derived from W-sperm disappeared during the anaphase of the first cleavage. The developmental rate was higher in triploid embryos polyploidized with heat shock, and lower in the gynogenetic haploid embryo than in the normal diploid embryos. In the gynogenetic diploid embryos, the metaphase of the first cleavage occurred at the same time as normal diploid embryos.
• Oogenesis and changes in the levels of reproductive hormones in triploid female rainbow trout

  T Kobayashi; S Fushiki; N Sakai; A Hara; M Amano; K Aida; M Nakamura; Y Nagahama

  FISHERIES SCIENCE JAPAN SOC SCI FISHERIES SCI 64 (2) 206 - 215 0919-9268 1998/04 [Refereed]
   

  The reproduction of artificially induced triploid rainbow trout was histologically and endocrinologically examined during three consecutive seasons. The gonadosomatic indices of triploid females were much lower (0.02-0.06) than the indices of diploid females. The concentrations of sex steroid hormones, including testosterone, estradiol-17 beta, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, were much lower than those of diploid females but had small changes, and changes of gonadotropin (GtH) levels in the serum of triploid females differed from those of diploid females. Oogenesis in the triploid ovary was substantially delayed, and most germ cells ceased to develop at the synaptic stage of the first meiotic division. At three years old, various developmental stages of oocytes were detected in the ovary, such as peri-nucleolus, yolk vesicle, and early yolk globule stage. It seemed that these rare developments of triploid oocytes were related to the small increases of steroid hormone concentrations and rise of GtH level that appeared when fish were two years old and three years old. Nevertheless, even in triploid ovarian oocytes grown to secondary yolk globule stage, the germinal vesicles did not migrate and there was no ovulation. It was confirmed that triploid liver cells possessed the potential to produce vitellogenin. These results suggest that the genital gland of triploid females is undeveloped because the odd number of chromosome sets causes the first maturation division to fail, and the endocrinological abnormality.
• Application of Fluoro-RAPD PCR analysis using Automate DNA sequencer

  Toru Kobayashi; Seiji Ohno; Takanori Kobayashi

  DNA多型 6 219 - 226 1998 [Refereed]
  
• 耐病性育種，7章 育種と耐病性，魚類防疫－魚病と人間の関わり－

  小林 徹

  海洋 海洋出版 号外14 (14) 206 - 210 0916-8575 1998
• The Competition for Food between Triploids and Diploids and its Effect on the Growth of Triploids in Rainbow Trout

  Toru Kobayashi; Shozo Fushiki

  SUISANZOSHOKU 45 (1) 87 - 96 0371-4217 1997/03 [Refereed]
  
• Survival and cytological observations on early development of normal, hybrid, and gynogenetic embryos of amago salmon

  T Kobayashi

  FISHERIES SCIENCE JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES 63 (1) 33 - 36 0919-9268 1997/02 [Refereed]
   

  The artificial induction of gynogenesis of amago salmon was cytologically confirmed, and the nucleus behavior and the cytological rates were compared at the early stage of development among the following three groups: normal, hybrid between the female amago salmon and the male rainbow trout, and gynogenetic developed group. The first cleavage of hybrid and gynogenetic development was delayed compared with normal development, while that in the gynogens was later than that of the hybrid. The cytological stage at 68 h. degrees C in cumulative temperature, which is considered the optimum timing for suppressing the first cleavage of gynogen, was prometaphase. In gynogenetically activated eggs, the lump-shaped body appeared in the metaphase plate. Then, it remained near the cleavage plane in the following cell division, and was considered to be degenerated nuclear materials derived from irradiated sperm, i.e. dense chromatin body (DCB), which could not transform to chromosomes. Therefore, paternally derived genetic materials should disappear during the anaphase and the telophase in the first cleavage of gynogenetic development. Some chromosomes were gradually eliminated in the hybrid development after the first cleavage. This phenomenon was considered a major cause of inviability and the subsequent expression of abnormality during the development of the hybrid.
• 染色体操作によるサケ科魚類の三倍体および雌性発生二倍体の生物学的特性並びに形質の遺伝に関する研究

  小林 徹

  東京大学農学生命科学研究科審査博士学位論文 PP 304 - 230 1997
• Oxygen Uptake Rate and Opercular Movement at Resting and Recovery from Exhaustive Exercise of Treploid Amago Salmon, Oncorhynchus rhodurus

  Toru Kobayashi; Katsutoshi Hori; Shozo Fushiki; Soichi Nakamura; Koichi Ueno

  SUISANZOSHOKU 水産増殖談話会 43 (3) 395 - 400 0371-4217 1995/09 [Refereed]
  
• Importance of a Reed Community as a Nursery for Nigorobuna, Carassius auratus grandoculis.

  Kohichi Fujiwara; Takahiro Usui; Toru Kobayashi; Eiji Mizutani

  Kankyo System Kenkyuu 23 414 - 419 1995 [Refereed]
  
• Production of cloned Amago salmon Onchorhynchus rhodurus

  T KOBAYASHI; A IDE; T HIASA; S FUSHIKI; K UENO

  FISHERIES SCIENCE JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES 60 (3) 275 - 281 0919-9268 1994/06 [Refereed]
   

  We induced homozygous clones of amago salmon by gynogenetic chromosome manipulation. Mitotic gynogenesis diploid (mitotic-G2N) as first generation was produced by suppression of the first cleavage of the eggs inseminated with UV-irradiated sperm of rainbow trout by hydrostatic pressure shock. The yield of apparently normal gynogenetic embryos was 2.26% when the eggs were shocked at 650 kg/cm2 of hydrostatic pressure for 6 min at 5 h 30 min (approximately 68 h . degrees-C in cumulative temperature) after insemination. Homozygous evidence of gynogenetic juveniles was demonstrated by the segregation of Idh-3 and Pgm-1 loci. Five homozygous inbred strains were produced from the eggs of homozygous gynogenetic females by retaining the second polarbody, while outbred controls were produced for each strain by crossing with males of other strains. The clonal proof of these strains was demonstrated by the immunological acceptance of operculum allografts from inbred sisters. Similar survivals were shown among clones produced by mothers from identical strain. On the other hand, various survivals were seen between the clones using maternal parents from different strains. These results suggested that the line selection was a vital factor in clone production.
• TESTICULAR DEVELOPMENT AND CHANGES IN THE LEVELS OF REPRODUCTIVE HORMONES IN TRIPLOID MALE RAINBOW-TROUT .2. STUDY OF BIOLOGICAL CHARACTERS AND THE GENETICS OF SOME TRAITS OF TRIPLOID AND GYNOGENETIC DIPLOID ON SALMONID FISHES

  T KOBAYASHI; N SAKAI; S FUSHIKI; Y NAGAHAMA; M AMANO; K AIDA

  NIPPON SUISAN GAKKAISHI JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES 59 (6) 981 - 989 0021-5392 1993/06 [Refereed]
   

  Testicular development and hormone levels of triploid males of rainbow trout were compared with those of diploid males during three consecutive seasons. Changes in the levels of serum sex steroid hormones (testosterone, 11-ketotestosterone, and 17alpha,20beta-dihydroxy-4-pregnen-3-one) and pituitary and serum gonadotropin in triploids were similar to those in diploids. Although meiotic division first occurred in the July testes of both triploids and diploids, subsequent spermatogenesis was markedly delayed in the testes of triploids. During the natural spermiating season, a large number of atretic figures of spermatogenic germ cells were found in the testes of triploids with a sudden decrease in the gonadosomatic index. The amount of sperm was markedly less in triploids than in diploids. Some necrotic cells were invested by hypertrophied Sertoli cells lining the inner wall of the lobular lumen. Taken together, these results suggest that the cause of abnormality of spermatogenesis in triploids is the disruption of meiosis due to the odd number of chromosome sets.
• Changes in growth hormone and prolactin messenger ribonucleic acid levels during seawater adaptation of Amago salmon (Onchorhynchus rhodurus)

  T Yada; T Kobayashi; A Urano; T Hirano

  JOURNAL OF EXPERIMENTAL ZOOLOGY WILEY-LISS 262 (4) 420 - 425 0022-104X 1992/07 [Refereed]
   

  To examine the changes in secretion of growth hormone (GH) and prolactin (PRL) with reference to their osmoregulatory roles, changes in pituitary mRNA levels and plasma concentrations of these hormones were examined during seawater adaptation in silvery juveniles (smolts) and precociously mature mates (dark parr) of amago salmon (Oncorhynchus rhodurus). Transfer to seawater increased plasma sodium levels in both smolts and dark parr. Smolts adjusted their plasma sodium to the level associated with seawater-adaptation (165 mEq/liter) within 3 days, whereas no adjustment was seen in dark parr; the latter failed to survive in seawater for more than 3 days. In smolts, plasma GH levels increased significantly 1 day after transfer, whereas there was no significant change in dark parr. An increase in GH mRNA levels was observed in smolts in association with increased plasma GH, whereas there was no change in dark parr. In contrast, a reduction in plasma PRL levels was consistently observed in both smolts and dark parr after transfer to seawater. However, there was no significant change in PRL mRNA levels in either smolts or dark parr. These results suggest that both gene expression and release of GH are activated by seawater transfer only in smolts with adequate seawater adaptability, whereas PRL gene expression is decreased after seawater transfer regardless of seawater adaptability.
• Growth, Survival and Reproductive Cycle of Induced Triploid Rainbow Trout under the Communal Rearing Condition with Diploid for Long Period.

  Toru Kobayashi

  SUISANZOSHOKU 40 (1) 57 - 70 0371-4217 1992/03 [Refereed]
  
• ニジマス・アマゴにおける雌性発生法の育種への応用とクローン作出

  小林 徹

  水産育種 17 1 - 4 1991

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