Thermally selected male rainbow trout selectively bred at high temperatures were crossed with females of the Nikko strain with normal thermal tolerance. The F2 pedigrees, comprising 1641 individuals, were divided into 26 batches. The fish were acclimated to 17 degrees C, then their thermal tolerance determined by measuring the effective time (ET) required for them to lose equilibrium at an approximate upper lethal temperature (28 degrees C). The fish were categorized into 0- to < 20-, 20- to < 40-, 40- to < 60-, and <= 60-min ET groups. The individuals that survived the first treatment were subjected to a second experiment to determine the ET again for their respective groups. High reproducibility, i.e., 100%, for the 0- to < 20-min ET group was observed in the third experiment for individuals grouped into this ET category in the second experiment; 85.5% of individuals classified into the <= 60-min ET in the first experiment were recovered in the second experiment. Thus, our findings demonstrate that ET is a useful indicator for discriminating phenotypes according to their thermal tolerance.

A thermally selected strain of rainbow trout has been established by selective breeding since 1966 in Miyazaki, Japan. In the present study, we compared the critical thermal maxima (CTMs), the temperatures at which organisms reach a predefined sublethal endpoint and lose their equilibrium, between a thermally selected and two normal (Donaldson) strains of rainbow trout. The CTM of one normal strain from Nikko (Nikko strain) acclimated to 20 °C (29.7 °C) was significantly lower than those of the thermally selected strain (30.0 °C) and the other Donaldson strain from Aomori (29.9 °C) (P < 0.05). The F1 generations, F1T and F1N, were produced by crossing thermally selected strain females with Nikko strain males and Nikko strain females with thermally selected strain males, respectively. No significant difference was observed in the CTM between F1T [30.1 ± 0.15 °C (n = 30)] and F1N [30.1 ± 0.16 °C (n = 30)] (P > 0.05) for fish acclimated to 20 °C, suggesting that the F1 offspring inherited the thermal tolerance trait from one thermally selected strain parent irrespective of whether it was the male or female. F2 offspring of F1T or F1N also showed the thermal tolerance trait. The coefficients of variation for CTM were also compared among all the datasets obtained in the present study and their values for F1 hybrids were lower than those of the parental generation of the Nikko strain (P < 0.05). In contrast, the coefficients of variation of F2s were the same as those of their parental generation. Furthermore, the thermally selected strain and Nikko strain as a reference family provide a F2 generation for segregating phenotypes, which is required for in-depth genetic analysis of the thermally selected rainbow trout strain.

Rainbow trout Oncorhynchus mykiss is a cold-water aquaculture species, and a thermally selected strain has been developed by multigenerational high-temperature breeding in Japan. We examined the expression of heat-shock proteins as candidates responsible for thermotolerance in rainbow trout using F2 offspring from F1 hybrids produced between thermally selected and normal strains. From F2 offspring, two groups were selected for western blot analysis, namely, low- and high-thermotolerance groups (times to loss of equilibrium were < 30 and a parts per thousand yen60 min, respectively). We demonstrated that the expression levels of Hsp70, Hsp60, and Hsp40 in tail fin tissues were significantly higher in the individuals with high thermotolerance than in those with low thermotolerance under non-heat-shock conditions. In particular, Hsp70 was expressed only in the individuals with high thermotolerance. These results suggest that Hsp70 is a major protein responsible for conferring thermotolerance in rainbow trout.

The objective of the study reported here was to investigate genes related to upper temperature tolerance in rainbow trout Oncorhynchus mykiss, a cold-water species with considerable economic importance, by global gene expression analysis using a next generation sequencing system. Fifty million paired sequences were collected from the gill tissues of each of five individuals of a thermally selected strain developed by selective breeding and from the gill tissues of a standard Donaldson strain and assembled into transcripts. The data of both strains were integrated, and a BLASTX search identified 13,092 independent, known genes. A back-mapping of raw reads from both strains onto the genes, conducted to investigate their frequency of expression, revealed that 324 genes showed at least a twofold higher expression in the thermally selected strain than in the Donaldson strain. In addition, 44.4 % of commonly expressed genes were categorized into 38 functional groups by annotation. Genes encoding heat shock proteins and c-fos-related proteins were highly expressed in the thermally selected strain. Our strategy to employ next generation sequencing proved to be very useful to find genes responsible for upper temperature tolerance of rainbow trout.

Feeding activity was examined at high temperatures by using a demand feeder for thermally selected and normal rainbow trout Oncorhynchus mykiss strains. When the water temperature was raised in experiment 1 from 17.5 to 25.7 degrees C at 0.3 degrees C/day in 21 days, the average daily food consumption rate in the thermally selected strain rose to 7.1%, which was significantly higher than that of the normal strain (4.1%, P < 0.05). The corresponding rate was also significantly higher in the thermally selected (0.8%) than in the normal strain (0.2%, P < 0.05) of fish in experiment 2 where water temperature was raised to 24.1 degrees C in 0.5 degrees C/day increments. When water temperature was raised rapidly in experiment 3 from 16.7 to 21.7 degrees C in one day and gradually to 24.4 degrees C in 28 days at 0.1 degrees C/day, the average daily food consumption rates were 1.0 and 0.1% for the thermally selected and normal strains, respectively, with significant difference (P < 0.01). These results suggest that the thermally selected strain has acquired thermal tolerance as a result of artificial selection.

To investigate molecular mechanisms involved in thermal resistance of rainbow trout, Oncorhynchus mykiss, embryos from thermally selected strain in various developmental stages were treated at 22 degrees C for 30 min and subsequently developed at 12 degrees C using the Donaldson strain as a reference. The embryos were evaluated for their hatching rate along with the ratio of embryos having an abnormal appearance and subjected to mRNA arbitrarily primed reverse transcription-polymerase chain reaction (RAP RT-PCR). One of the genes dominantly expressed in the thermally selected strain (COX II) coded for cytochrome c oxidase subunit II. Northern blot analysis revealed that the accumulated levels of COX II transcripts were more abundant in embryos and unfertilized eggs from the thermally selected strain than those from the Donaldson strain. Furthermore, the differential expression patterns of the ATPase 6-8 gene were similar to those of the COX II gene, whereas the ATP synthase beta-subunit gene showed no significant differences between the two strains.

Thermal tolerance was studied in a rainbow trout strain successively selected through high temperature breeding at 20-2 degrees C since 1966 in Miyazaki Prefecture, Japan. The hatching rate and fry mortality at high temperatures were examined in the selected strain along with normal strains cultured at water temperature of 9-17 degrees C. The hatching rate of embryos fertilized at either 10 or 14 C and subsequently subjected to high temperatures in the blastula or neurula stage of the selected strain, was marginally higher than that of the normal strain counterparts. The upper 50% lethal temperatures (LT50) for embryos in the early segmentation, blastula and neurula stages of the selected strain were also higher than those of the normal strain counterparts. Death temperatures and LT50 of fries acclimated to 20 degrees C of the selected strain were significantly higher than those of the normal strains. However, no difference in the critical thermal maximum was detected between the different strains. These results suggest that the selected strain of rainbow trout established by selecting successively for many generations at high temperatures acquired a degree of thermal tolerance.

A method of introducing protease inhibitors into fish muscle through the bulbus arteriosus was developed using an in situ perfusion technique. Perfusion efficiency was initially tested using eosin and [S-35]-methionine. Visible fluorescence was observed in the gill, liver, intestine and dorsal muscle of the eosin-treated tilapia, and the occurrence of eosin in the blood vessels of the dorsal muscle was confirmed under a fluorescence stereoscopic microscope with ultraviolet light. The radioactivity of [S-35]-methionine was taken into the dorsal muscle and liver at a concentration of 7.8 Bq/g and 70.2 Bq/g, respectively, after perfusion with 1000 Bq/mL solution. Using the perfusion technique with four kinds of protease inhibitors dissolved in physiological saline, the type of proteases implicated in the post-mortem muscle softening in tilapia (867 +/- 195 g, n = 10/protease inhibitor) was investigated. After the perfusion of leupeptin (serine and cysteine protease inhibitor), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk; caspase inhibitor), chymostatin (serine protease inhibitor) and o-phenanthroline (metalloprotease inhibitor), the breaking strength of the perfused muscle was measured as a parameter of the meat toughness and compared with that of the control fish, which were perfused with physiological saline only. The reduction of breaking strength during storage was inhibited by the perfusion of leupeptin and Z-VAD-fmk.

Immunomodulatory effects of immunoactive peptides from the fermented products of chicken egg (EF203) administered orally to rainbow trout, Oncorhynchus mykiss, were investigated. The chemiluminescent responses of kidney phagocytes after treatment with EF203 were significantly increased. Fish administered EF203 showed high phagocytic activities as compared to controls, and immunomodulatory effects were found to be dose dependent. Fish treated with EF203 displayed an increased resistance to both natural and experimental beta-haemolytic streptococcal infection.

分担者らの協力のもとに作出したクロマグロとスマの雑種個体の多型タイピングを行い、スマ、クロマグロの連鎖地図を作成した。さらに分担者から、ブリとヒラマサの雑種、ニホンウナギとオオウナギの雑種、アユの半数体、シイタケの単核相細胞、マツタケの単核相細胞の提供を受け、それらからのゲノムの抽出と、全ゲノムシーケンシングによる多型タイピングを進めており、一部完了した。またハンガリーの共同研究者からチョウザメとヘラチョウザメの雑種の提供を受けて実験を進めている。 ナマズ、アユの半数体、雌性発生胚の作出を実施した。その結果、ナマズは半数体、雌性発生ともに受精率が極めて低かったためサンプルを得ることができなかった。アユは多数の半数体卵のサンプルを得ることができた。ニホンウナギとオオウナギの雑種は極めて高い受精率、ふ化率を示し、雑種胚及び雑種仔魚のサンプルを得ることができた。 植物に関して2020年度は新型コロナの影響があったが、2021年度は下記の実験を開始でき、以下の研究結果を獲得した。シイタケとマツタケの単核相細胞（一次菌糸または胞子）を準備し、連鎖解析を開始した結果、ドラフトゲノムの質を著しく改善できた。アカマツとクロマツの交雑個体（アイグロマツ）の作出を開始し、現時点で確定的ではないが受粉が成功していると期待できる結果を得ている。 それぞれの生物のエクソソーム解析を推進するための予備的な検討を行った。それぞれの種でタンパク質配列のデータベースが整備されていることが解析には必要であるため、ゲノム配列からそれらのデータベースの構築を試みている。 イトヨについてハプロイドゲノムタイピングのバリエーションとして個々の精子のシングルセルゲノム解読によるタイピングを検討し、連鎖地図を構築することができた。

DNAマーカー等のゲノム情報を活用し短期間で効率的に高温耐性などの優良な形質を評価し，家系選抜や交雑等により，温暖化の影響を回避できる養殖品種を開発する

DNAマーカー等のゲノム情報を活用し短期間で効率的に高温耐性などの優良な形質を評価し，家系選抜や交雑等により，温暖化の影響を回避できる養殖品種を開発する

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