Discrimination of amphidromous and landlocked ayu, Plecoglossus altivelis altivelis (Teleostei: Plecoglossidae) in the Nuta River, Hiroshima Prefecture, using the otolith Sr/Ca ratio

6～8月にかけて主に沼田川中流域で採集したアユ30尾の耳石Sr/Ca比を調べ，由来判別を行った。その結果，30尾中12尾が天然遡上アユであることが判明した。また，支流の椋梨ダム湖（白竜湖）より上流の椋梨川で採集した13尾は，全ての個体で耳石Sr/Ca比の変動が認められなかったことから，これらはダム湖で再生産した陸封アユと考えられた。Ayu, Plecoglossus altivelis altivelis, is a commercially important species of amphidromous fish. Thus it is being targeted for stock replenishment to sustain harvest numbers. Stock identification between amphidromous and hatchery fish was conducted using the otolith Sr/Ca concentration ratio on fish taken from the Nuta River in Hiroshima Prefecture, where 170,000 hatchery ayu had been previously released. Of 30 fish sampled from the middle-reaches, 12 were identified as an amphidromous form. Of the 13 fish sampled from the Mukunashi River, a branch of the Nuta River, all were identified as landlocked fish that would have completed their life cycle between the Mukunashi River and the artificial lakes formed by the Mukunashi Dam.本研究は平成19年度広島大学地域貢献研究において，東広島市沼田川漁業協同組合より課題提案された「東広島市沼田川におけるアユの資源保全と有効利用」の一環として行った

A sensitive non-radioactive in situ hybridization method for the detection of chicken IgG γ-chain mRNA: a technique suitable for detecting of variety of mRNAs in tissue sections

We established a sensitive non-radioactive in situ hybridization (ISH) method for the detection of chicken IgG γ-chain mRNA in paraffin sections. RNA probes were transcribed in vitro from cloned chicken IgG CH1 nucleotide sequences with SP6/T7 RNA polymerases in the presence of DIG-UTP. These probes were used for hybridization and were immunodetected using anti-DIG antibodies conjugated to horseradish peroxidase. The immunoreactive products were visualized with DAB-H(2)O(2). IgG γ-chain mRNA-expressing cells were localized in both the spleen and oviductal tissues. This method demonstrated an excellent sensitivity since the ISH signal was clear and the background was negligible. We found that in the spleen IgG γ-chain mRNA-expressing cells were present mainly in the red pulp, whereas in the oviduct they appeared mainly in the mucosal stroma and not in the mucosal epithelium

マスウキブクロセンチュウ(新称)Salvelinema salmonicolaの新宿主と新分布地

広島県西部を流れる太田川水系の筒賀川とその支流である猪股川においてアマゴ(Oncorhynchus masou ishikawae Jordan and McGregor)の鰾から線虫類のSalvelinema salmonicola (Ishii, 1916)を採集した。アマゴは本線虫の新宿主である。今回の採集によって, 北海道から滋賀県の範囲にあった我が国での本種の分布域は広島県にまで拡大した。本記録により, 広島県のサケ科魚類から記録された寄生虫は6種, 太田川水系のサケ科魚類から得られた寄生虫は3種となる。採集地におけるヨコエビ類を中間宿主とする本種の生活環について考察するとともに, 河川工事が終宿主や中間宿主, 本種に与える影響について論議した。本種とSalvelinema属の両者にマスウキブクロセンチュウ(鱒鰾線虫)の新標準和名を提唱する。The cystidicolid nematode Salvelinema salmonicola (Ishii, 1916) is a parasite of freshwater salmonids in the North Pacific rim region, including Japan, Far Eastern Russia, Alaska (USA) and northern British Columbia (Canada). This species was found in the swim bladder of river-resident amago salmon (Oncorhynchus masou ishikawae Jordan and McGregor)(Salmoniformes: Salmonidae) from the Tsutsuga River and its tributary, Inomata River, of the Ota River System in Hiroshima Prefecture, western Honshu, Japan. This is the first record of S. salmonicola from western Japan, extending its distribution from central to western Honshu in the country. Oncorhynchus masou ishikawae is a new host for S. salmonicola. The life cycle of S. salmonicola and the impact of the past and current construction on the fish definitive host, invertebrate (probably amphipod) intermediate host and parasite populations in the sampling locality are discussed

胚中心におけるT細胞セレクション機構の解析

研究期間:平成11-12年度 ; 研究種目: 基盤研究C2 ; 課題番号:1167032

Expression of ayu antimicrobial peptide genes after LPS stimulation

Plecoglossus altivelis (ayu) is one of the most important fish species in the Japanese islands and in internal fish hatcheries. Living in open aquatic environments exposes fish to many pathogens. Therefore, they require rapid and strong immune defenses. We investigated in vivo the direct association between the ayu innate immune response, represented by the relative transcription of genes encoding the cathelicidin and hepcidin antimicrobial peptides, and lipopolysaccharide (LPS), a conventional pathogen-associated molecular patterns (PAMPs) of Gram-negative bacteria. Different concentrations of LPS (1, 10 and 100 µg/fish) were injected intraperitoneally into young (sexually immature) and adult (fully sexually mature) ayu. The relative expression of the antimicrobial peptide genes was measured 6 hr, 24 hr and 1 week after stimulation with LPS. We found a direct association between the expression of the antimicrobial peptide genes investigated and LPS stimulation. This relationship was time-, dose- and age-dependent. Further research is required to determine the cell-specific transcriptional regulation and posttranscriptional regulation of these antimicrobial peptides

Studies on Morphology and Cytochemistry in Blood Cells of Ayu Plecoglossus altivelis altivelis

Peripheral blood cells from ayu, Plecoglossus altivelis altivelis, were separated using a density gradient. Blood cells were then smeared using Shandon Cytospin and subjected to cytochemical staining. Blood cells were categorized based on morphological and cytochemical characteristics, and the density fractionation range and nucleus area/cell area ratio were observed. Lymphocytes are distinguished from neutrophils by their basophilic cytoplasm and Golgi-like field. The features of chromatin in thrombocytes are different from those of lymphocytes or neutrophils, but some small neutrophils have similar chromatin. Therefore, it is necessary to perform peroxidase staining to distinguish small neutrophils from thrombocytes. Basophils have large basophilic granules in cytoplasm. Based on density fractionation of blood cells, thrombocytes in the low-density area were separated from other blood cells. Identification of peripheral blood cells from ayu was possible with these staining methods. Monocytes/macrophages from spleen are specifically positive for esterase staining by α-naphthyl butyrate. As a result, thrombocytes, lymphocytes, neutrophils, basophils and monocytes/macrophages were identified in smears from peripheral blood or spleen tissue. In this paper, we confirmed that the peripheral blood corpuscles of ayu are able to be identified using the present staining methods

An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors

At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing.This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers 19H03107, and 19K22286