JP2019154367A - Transsexual induction method of fishes, transsexual induction device of fishes and sex control method of fishes - Google Patents
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Abstract
Description
本発明は、魚類の性転換誘導方法、魚類の性転換誘導装置及び魚類の性統御方法に関する。 The present invention relates to a fish sex change induction method, a fish sex change induction device, and a fish sex control method.
商取引される魚類には、性によって商品価値が異なる魚種がある。例えば、トラフグでは、オスの精巣に価値があり、シロザケではメスの卵巣にある魚卵に価値がある。ヒラメ等では、オスとメスとで成長に差があり、成長が早いメスが養殖業者に好まれる。したがって、魚類養殖業ではオス又はメスを優先的に生産する技術が求められている。 There are fish species that have different commercial values depending on their sex. For example, in the tiger puffer, male testes are valuable, and in chum salmon, the fish eggs in the female ovaries are valuable. In flounder and the like, there is a difference in growth between males and females, and fast-growing females are preferred by farmers. Therefore, a technique for preferentially producing males or females is required in the fish farming industry.
既に染色体操作、遺伝子操作、早期性判別、性ホルモン投与及び環境操作といった性統御を行う技術が開発されている。しかし、染色体操作及び早期性判別は、世代をつなぐため時間がかかる。さらに遺伝子操作自体が技術的な困難さを伴う。一方、性ホルモンの投与は魚体内への残留が懸念され、消費者に対して不安を与えることがある。さらに、性ホルモンの注射での投与は煩雑で、稚魚及び小型の魚類に対する注射での投与は難しい。 Techniques for sex control such as chromosome manipulation, genetic manipulation, early sex discrimination, sex hormone administration and environmental manipulation have already been developed. However, chromosome manipulation and early sex discrimination take time to connect generations. Furthermore, genetic manipulation itself involves technical difficulties. On the other hand, the administration of sex hormones is feared to remain in the fish body, which may give anxiety to consumers. Furthermore, administration by sex hormone injection is complicated, and administration by injection to fry and small fish is difficult.
環境操作では、難しい技術は求められず、性ホルモンの残留等による悪印象を消費者に与えることを避けられる。魚類の性分化は、遺伝的要因と環境要因とによって決定される。性分化に影響を与える環境要因として、水温、ｐＨ及び飼育密度が知られている。 Environmental manipulation does not require difficult skills and avoids giving consumers a bad impression of residual sex hormones. Sexual differentiation of fish is determined by genetic and environmental factors. Water temperature, pH, and breeding density are known as environmental factors that affect sexual differentiation.
性分化に影響を与える水温は通常の飼育温度より高温である。高い水温を維持するのにはコストがかかる。ｐＨ及び飼育密度の調整も煩雑で、特に大型の魚類であれば技術的に困難となる。 The water temperature that affects sexual differentiation is higher than normal breeding temperature. It is expensive to maintain a high water temperature. Adjustment of pH and breeding density is also complicated, and technically difficult especially for large fish.
簡便、かつ比較的低コストで性転換を誘導する方法として光を利用する方法がある。特許文献１には、マツカワの孵化後、性が決定されるまでの間に赤色光のみを所定期間照射することにより、メス化を促進させることができることが開示されている。 There is a method using light as a method for inducing sex change at a simple and relatively low cost. Patent Document 1 discloses that feminization can be promoted by irradiating only red light for a predetermined period after Matsukawa hatching until sex is determined.
上記特許文献１に開示されたメス化を促進させる性統御方法では、赤色光を照射されたすべてのマツカワがメスになるわけではない。赤色光を照射されたマツカワにおけるメスの割合は最大で５９．５％であった。 In the sex control method that promotes feminization disclosed in Patent Document 1, not all Matsukawa irradiated with red light becomes female. The maximum percentage of females in Matsukawa irradiated with red light was 59.5%.
遺伝的性がメス、すなわち性染色体がＸＸ型であるにも関わらず精巣を有する偽オスが得られれば、偽オスとメスとを交配することで、次世代がすべてメスとなる全メス化が実現できる。したがって、遺伝的性がメスである魚類のオス化を簡便かつ低コストで誘導することができる技術が求められている。 If genetic sex is female, that is, a pseudomale with testis is obtained despite the sex chromosome being XX type, mating the pseudomale with a female, the next generation can be all female realizable. Therefore, there is a need for a technique that can easily and inexpensively induce maleization of fish whose genetic sex is female.
本発明は、上記実情に鑑みてなされたものであり、遺伝的性がメスである魚類のオス化を簡便かつ低コストで誘導することができる魚類の性転換誘導方法及び魚類の性転換誘導装置、並びに簡便かつ低コストで全メス化が可能な魚類の性統御方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and is a fish sex change induction method and fish sex change induction apparatus capable of easily and inexpensively inducing maleization of fish whose genetic sex is female. Another object of the present invention is to provide a sex control method for fish that is feasible and can be made completely female at a low cost.
本発明の第１の観点に係る魚類の性転換誘導方法は、
孵化後の魚類に対して、性が決定されるまでに緑色光を照射する照射ステップを含む。
The fish sex change induction method according to the first aspect of the present invention comprises:
An irradiation step of irradiating the fish after hatching with green light until sex is determined is included.
この場合、前記緑色光は、
５１３〜５２３ｎｍに波長のピークを有する、
こととしてもよい。
In this case, the green light is
Having a wavelength peak at 513-523 nm,
It is good as well.
また、前記魚類は、
性染色体で性が規定される魚類である、
こととしてもよい。
In addition, the fish
A fish whose sex is regulated by sex chromosomes.
It is good as well.
また、前記魚類は、
性染色体で規定される性がメスである、
こととしてもよい。
In addition, the fish
The sex defined by the sex chromosome is female,
It is good as well.
本発明の第２の観点に係る魚類の性転換誘導装置は、
魚類に対して緑色光を照射する光源を備える。
The fish sex change induction device according to the second aspect of the present invention is:
A light source for irradiating fish with green light is provided.
本発明の第３の観点に係る魚類の性統御方法は、
孵化後の魚類に対して、性が決定されるまでに緑色光を照射する照射ステップと、
前記照射ステップにおいて前記緑色光を照射された、性染色体で規定される性がメスである魚類が有する精子により、該魚類の未受精卵を受精させる受精ステップと、
を含む。
The fish sex control method according to the third aspect of the present invention includes:
An irradiation step of irradiating the fish after hatching with green light until sex is determined;
A fertilization step of fertilizing an unfertilized egg of the fish by a sperm of a fish whose sex defined by a sex chromosome is irradiated with the green light in the irradiation step;
including.
本発明によれば、遺伝的性がメスである魚類のオス化を簡便かつ低コストで誘導することができる。さらに、簡便かつ低コストで全メス化が可能になる。 ADVANTAGE OF THE INVENTION According to this invention, maleization of the fish whose genetic sex is a female can be induced | guided | derived simply and at low cost. Furthermore, it becomes possible to make all females easily and at low cost.
本発明に係る実施の形態について図面を参照しながら説明する。なお、本発明は下記の実施の形態によって限定されるものではない。 Embodiments according to the present invention will be described with reference to the drawings. In addition, this invention is not limited by the following embodiment.
（実施の形態１）
本実施の形態に係る魚類の性転換誘導方法は、孵化後の魚類に対して、性が決定されるまでに緑色光を照射する照射ステップを含む。孵化直後から又は孵化後２４時間以内の稚魚に緑色光を照射するのが好ましい。「性が決定される」とは、具体的には、性分化の結果、精巣又は卵巣を有することをいう。以下では、精巣又は卵巣を有することで判別される性を「表現型の性」とする。
(Embodiment 1)
The fish sex change induction method according to the present embodiment includes an irradiation step of irradiating the fish after hatching with green light until sex is determined. It is preferable to irradiate green light immediately after hatching or to fry within 24 hours after hatching. “Gender is determined” specifically means having sex or ovaries as a result of sex differentiation. Hereinafter, sex determined by having testis or ovary is referred to as “phenotypic sex”.
表現型の性は、生殖腺の組織を観察することで判別できる。精巣を有する場合、表現型の性はオスである。卵巣を有する場合、表現型の性はメスである。性が決定されるまでの期間は魚種によって様々であるため、緑色光を照射する期間は魚種に応じて適宜設定すればよい。緑色光を照射する期間は、例えば、１〜４週間、１か月、２か月、３か月、４か月又は６か月である。 Phenotypic sex can be determined by observing gonad tissue. When having a testis, the phenotypic sex is male. When having an ovary, the phenotypic gender is female. Since the period until sex is determined varies depending on the fish species, the period during which the green light is irradiated may be appropriately set according to the fish species. The period of green light irradiation is, for example, 1 to 4 weeks, 1 month, 2 months, 3 months, 4 months, or 6 months.
照射ステップでは、魚類に対して間欠的に緑色光を照射してもよい。例えば、一定時間の明期と一定時間の暗期とを繰り返す明暗周期下で魚類を飼育する場合、緑色光は、明期に照射してもよい。 In the irradiation step, the fish may be irradiated with green light intermittently. For example, when raising fish under a light / dark cycle in which a light period of a certain time and a dark period of a certain time are repeated, the green light may be irradiated during the light period.
魚類に照射する緑色光の波長は、４９５〜５７０ｎｍである。好ましくは、緑色光は５１３〜５２３ｎｍに波長のピークを有する。好適には、緑色光は５１８ｎｍに波長のピークを有する。緑色光の放射照度は、任意であるが、例えば、０．５〜１００、０．５〜５０又は１〜１０Ｗ／ｍ２である。 The wavelength of green light irradiated to fish is 495 to 570 nm. Preferably, the green light has a wavelength peak at 513 to 523 nm. Preferably, the green light has a wavelength peak at 518 nm. Although the irradiance of green light is arbitrary, it is 0.5-100, 0.5-50, or 1-10 W / m < 2 >, for example.
上記の魚類は、好ましくは性染色体で性が規定される魚類である。性染色体で性が規定される魚類は、メスの性染色体がＸＸ型でオスの性染色体がＸＹ型であるオスヘテロ型の魚類であってもよいし、メスの性染色体がＺＷ型でオスの性染色体がＺＺ型であるメスヘテロ型の魚類であってもよい。性染色体で規定される性を、遺伝的性という。遺伝的性は、例えば、性決定遺伝子の有無で判別できる。性決定遺伝子の有無は、ゲノムＤＮＡの塩基配列決定、好ましくは、ＰＣＲによって決定できる。 The fish is preferably a fish whose sex is defined by a sex chromosome. The fish whose sex is defined by the sex chromosome may be a male heterozygous fish in which the female sex chromosome is XX type and the male sex chromosome is XY type, or the female sex chromosome is ZW type and male sex. It may be a female hetero fish whose chromosome is ZZ. Sex defined by sex chromosomes is called genetic sex. Genetic sex can be determined, for example, by the presence or absence of a sex-determining gene. The presence or absence of a sex-determining gene can be determined by determining the base sequence of genomic DNA, preferably by PCR.
下記実施例に示すように、当該性転換誘導方法によれば、遺伝的性がメスである個体が、精巣を有する偽オスに誘導される。一方、遺伝的性がオスである個体に関しては性転換が誘導されない。よって、緑色光が照射される魚類は、遺伝的性がメスであることが好ましい。 As shown in the following Examples, according to the sex change induction method, an individual whose genetic sex is female is induced to a pseudo male having a testis. On the other hand, sex reversion is not induced in individuals whose genetic sex is male. Therefore, it is preferable that the fish irradiated with green light have a female genetic sex.
緑色光が照射される魚類としては、例えば、魚卵に価値がある魚類が好ましい。当該魚類としては、サケ（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｋｅｔａ）、ベニザケ（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｎｅｒｋａ）、ギンザケ（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｋｉｓｕｔｃｈ）、カラフトマス（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｇｏｒｂｕｓｃｈａ）、サクラマス（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｍａｓｏｕ）、マスノスケ（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｔｓｈａｗｙｔｓｃｈａ）、ニジマス（Ｏｎｃｏｒｈｙｎｃｈｕｓ ｍｙｋｉｓｓ）、イトウ（Ｈｕｃｈｏ ｐｅｒｒｙｉ）、イワナ（Ｓａｌｖｅｌｉｎｕｓ ｌｅｕｃｏｍａｅｎｉｓ）及びタイセイヨウサケ（Ｓａｌｍｏ ｓａｌａｒ）等のサケ科（Ｓａｌｍｏｎｉｄａｅ）の魚類の他、チョウザメ（Ａｃｉｐｅｎｓｅｒｉｄａｅ ｂｏｎａｐａｒｔｅ）及びシシャモ（Ｓｐｉｒｉｎｃｈｕｓ ｌａｎｃｅｏｌａｔｕｓ）等が挙げられる。 As the fish irradiated with green light, for example, fish that is valuable for fish eggs is preferable. As the fish, salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka), coho salmon (Oncorhynchus kisutch), pink salmon (Oncorhynchus gorbuscha), masu salmon (Oncorhynchus masou), chinook salmon (Oncorhynchus tshawytscha), rainbow trout (Oncorhynchus mykiss), Ito (Hucho perryi, salmoninus saluko, salmonid salmon (Salmonidae) and other species, as well as sturgeon (Acipenseridae bonaparte) and spruce (Spirinchus) anceolatus), and the like.
また、魚類としては、ヒラメ（Ｐａｒａｌｉｃｈｔｈｙｓ ｏｌｉｖａｃｅｕｓ）及びイシガレイ（Ｂａｓｉｌｅｗｓｋｙ）等の異体類が好ましい。異体類では、メスの成長がオスよりも早いため、全メス化できれば効率の良い養殖が可能となる。 In addition, as fish, foreign species such as flounder (Pararichthys olivaceus) and Ishigarei (Basilewsky) are preferable. In allogeneic species, females grow faster than males, so if all females are made, efficient aquaculture is possible.
ここで、本実施の形態に係る魚類の性転換誘導方法に好適な魚類の性転換誘導装置１００について説明する。図１は、性転換誘導装置１００の構成を示す図である。性転換誘導装置１００は、水槽１と、光源２とを備える。 Here, the fish sex change induction apparatus 100 suitable for the fish sex change induction method according to the present embodiment will be described. FIG. 1 is a diagram illustrating a configuration of the sex change induction device 100. The sex change induction device 100 includes a water tank 1 and a light source 2.
水槽１は、水又は海水等の飼育水とともに魚類を保持する。水槽１は、魚類を飼育できるものであれば形状等は特に限定されない。 The aquarium 1 holds fish together with breeding water such as water or seawater. The shape and the like of the aquarium 1 are not particularly limited as long as fish can be raised.
光源２は、水槽１の上に設置される。光源２は、魚類に対して上記の緑色光を照射する。光源２は、緑色光を発光するものであれば特に限定されないが、好ましくは緑色発光ダイオードである。 The light source 2 is installed on the water tank 1. The light source 2 irradiates the fish with the green light. The light source 2 is not particularly limited as long as it emits green light, but is preferably a green light emitting diode.
水槽１内には、送気のためのエアストーン３が設置される。エアストーン３は、チューブを介して送気装置と接続している。エアストーン３は、チューブを介して送られる空気を、水槽１内で放出する。エアストーン３が放出する空気によって、水槽１内の飼育水の酸素量が適切に維持される。 An air stone 3 for supplying air is installed in the water tank 1. The air stone 3 is connected to an air supply device via a tube. The air stone 3 releases the air sent through the tube in the water tank 1. The amount of oxygen in the breeding water in the aquarium 1 is appropriately maintained by the air released by the air stone 3.
水槽１内には、排水装置４が配設される。排水装置４は、サイフォン式で水槽１内の飼育水を水槽１の外部に適宜排出する。排水装置４によって、水槽１内の水位が所定の高さに維持される。 A drainage device 4 is disposed in the water tank 1. The drainage device 4 is a siphon type and appropriately drains the breeding water in the water tank 1 to the outside of the water tank 1. The water level in the water tank 1 is maintained at a predetermined height by the drainage device 4.
水槽１には、外部に設置された水槽５から飼育水が供給される。水槽５には、ヒーター６と、ポンプ７が配置される。水槽５には飼育水が貯められる。水槽５内の飼育水は、ヒーター６によって水槽１で飼育される魚類に適した温度に維持される。水槽５内の飼育水は、ポンプ７によってチューブを介して水槽１に送られる。 Breeding water is supplied to the aquarium 1 from an aquarium 5 installed outside. A heater 6 and a pump 7 are disposed in the water tank 5. Breeding water is stored in the aquarium 5. The breeding water in the aquarium 5 is maintained at a temperature suitable for fish bred in the aquarium 1 by the heater 6. The breeding water in the aquarium 5 is sent to the aquarium 1 through a tube by a pump 7.
以上詳細に説明したように、本実施の形態に係る魚類の性転換誘導方法によれば、簡便で低コストである緑色光の照射によって魚類のオスへの性転換を誘導することができる。 As explained in detail above, according to the fish sex change induction method according to the present embodiment, it is possible to induce sex change of fish to male by irradiation with green light which is simple and low cost.
また、緑色光が照射される魚類は、遺伝的性がメスであってもよいこととした。これにより、遺伝的性がオスである個体と遺伝的性がメスである個体とが水槽１に混在している場合よりも、水槽１で飼育された魚類において性転換が誘導される個体の割合を大きくすることができる。 In addition, fish that are irradiated with green light may be genetic in nature. Thus, the proportion of individuals in which sex change is induced in fish cultivated in aquarium 1, compared to the case where individuals with genetic sex are male and individuals with genetic sex are female Can be increased.
なお、照射ステップでは、緑色光のみが魚類に照射されてもよい。この場合、性転換誘導装置１００を遮光された暗室の内側又は外部から光が入射しない箱内に配置すればよい。 In the irradiation step, only green light may be irradiated to the fish. In this case, the sex change induction device 100 may be arranged in a box where light does not enter from the inside or outside of the dark room that is shielded from light.
（実施の形態２）
本実施の形態に係る魚類の性統御方法は、上記実施の形態１における照射ステップに加えて、受精ステップを含む。受精ステップでは、照射ステップにおいて緑色光を照射された、遺伝的性がメスである魚類が有する精子により、該魚類の未受精卵を受精させる。
(Embodiment 2)
The fish sex control method according to the present embodiment includes a fertilization step in addition to the irradiation step in the first embodiment. In the fertilization step, the unfertilized egg of the fish is fertilized by the sperm of the fish having a female genetic sex irradiated with the green light in the irradiation step.
魚類がヒラメ等のオスヘテロ型の場合、偽オスの性染色体はＸＸ型であるため、偽オスの精子にはＹ染色体は含まれず、Ｘ染色体が含まれる。したがって、偽オスの個体とメスである個体とを交配することで、上述のように次世代（Ｆ１世代）がすべてメスになる。魚類がイシガレイ等のメスヘテロ型の場合、当該性転換誘導方法によって得られる偽オスの性染色体はＺＷ型である。このため、偽オスの個体とメスである個体とを交配することで、性染色体がＷＷ型である超メスがＦ１世代に得られる。さらに、得られた超メスとオスである個体とを交配することで、Ｆ２世代をすべてメスにすることができる。 When the fish is of a male heterotype such as Japanese flounder, the pseudo-male sex chromosome is of the XX type, so the pseudo-male sperm does not contain the Y chromosome and contains the X chromosome. Therefore, by mating a pseudo male individual and a female individual, the next generation (F1 generation) becomes all female as described above. When the fish is a female hetero type such as flounder, the pseudo-male sex chromosome obtained by the sex change induction method is a ZW type. For this reason, a super female having a sex chromosome of WW type is obtained in the F1 generation by mating a pseudo male individual and a female individual. Furthermore, the F2 generation can all be turned into females by mating the obtained super female and male individuals.
受精ステップでは、偽オスの個体とメスの個体とを同じ水槽等で飼育することで自然に交配させればよい。また、受精ステップでは、メスから回収した未受精卵を偽オスから回収した精子に暴露することで人工的に受精させてもよい。 In the fertilization step, a pseudo male individual and a female individual may be bred naturally in the same aquarium or the like. In the fertilization step, an unfertilized egg collected from a female may be artificially fertilized by exposing it to sperm collected from a pseudo male.
以上詳細に説明したように、本実施の形態に係る魚類の性統御方法によれば、上記実施の形態１における照射ステップで得られる偽オスの精子を用いて未受精卵を受精させるので、簡便かつ低コストで、オスヘテロ型の魚類のＦ１世代及びメスヘテロ型の魚類のＦ２世代をより確実に全メス化することができる。 As described above in detail, according to the fish sex control method according to the present embodiment, an unfertilized egg is fertilized using a pseudo male sperm obtained in the irradiation step in the first embodiment. At low cost, the F1 generation of male hetero-type fish and the F2 generation of female hetero-type fish can be more reliably converted into all females.
なお、魚類がオスヘテロ型の場合、例えば、精子凍結技術を用いることで、必要な時に偽オスの精子を用いてＦ１世代を全メス化することができる。さらに、精子の選別技術を利用することでＦ１世代を全オス化することもできるため、魚種に応じてオス又はメスのどちらの性でも優占生産が可能になる。 When the fish is of a male hetero type, for example, by using a sperm freezing technique, the F1 generation can be made into all females using pseudo male sperm when necessary. Furthermore, since the F1 generation can be made completely male by using a sperm sorting technique, dominant production is possible for either male or female sex depending on the fish species.
なお、魚類がメスヘテロ型の場合、当該魚類の性統御方法は、照射ステップ及び受精ステップ（第１の受精ステップ）に加え、第１の受精ステップで得られた受精卵から孵化した超メスの未受精卵を、遺伝的性がオスである魚類が有する精子により受精させる第２の受精ステップを含むのが好ましい。この場合、当該性統御方法は、第１の受精ステップの後かつ第２の受精ステップの前に、第１の受精ステップで得られた受精卵から孵化した魚類の中から超メスを選抜する選抜ステップをさらに含んでもよい。 When the fish is a female heterozygous type, the sex control method for the fish is not applied to the ultra-female hatched from the fertilized egg obtained in the first fertilization step in addition to the irradiation step and the fertilization step (first fertilization step). It is preferable to include a second fertilization step in which the fertilized egg is fertilized by sperm possessed by a fish having a genetic sex. In this case, the sex control method is a selection method for selecting a super female from fish hatched from the fertilized egg obtained in the first fertilization step after the first fertilization step and before the second fertilization step. A step may be further included.
また、メスヘテロ型の魚類の性統御方法は、照射ステップの後かつ第１の受精ステップの前に、精子の選別技術により、遺伝的性がメスである魚類が有する精子の中からＷ染色体を含む精子を選別する選別ステップをさらに含んでもよい。こうすることで超メスを効率よく得ることができる。 The female heterosexual fish sex control method includes a W chromosome from sperm possessed by fish having a genetic sex by a sperm selection technique after the irradiation step and before the first fertilization step. A sorting step for sorting sperm may be further included. By doing so, a super knife can be obtained efficiently.
以下の実施例により、本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The following examples further illustrate the present invention, but the present invention is not limited to the examples.
（実施例１）
本実施例では、個体差を少なくするために近交系ＨｄＲｒ−II１系統のメダカ（Ｏｒｙｚｉａｓ ｌａｔｉｐｅｓ）を用いた。ＨｄＲｒ−II１系統では、体色を決定する遺伝子が性染色体上にあるため、体色と性とが対応している。オスの体色は黄色で、メスの体色は白色である。本実験に使用した親メダカＨｄＲｒ−II１系統は、ナショナルバイオリソースプロジェクトより入手した。
Example 1
In this example, inbred HdRr-II1 medaka (Oryzia latipes) was used to reduce individual differences. In the HdRr-II1 strain, the body color and the sex correspond to each other because the gene that determines the body color is on the sex chromosome. The male body color is yellow and the female body color is white. The parent medaka HdRr-II1 strain used in this experiment was obtained from the National BioResource Project.
市販されている３１．５×１８．５×２４．４ｃｍで約１２Ｌの水槽をメダカの飼育に用いた。光源には緑色光、白色光、赤色光又は青色光を照射するＬＥＤ（緑色光：ＬＢ１５２６Ｇ、白色光：ＬＤＡ６Ｎ−Ｇ、赤色光：ＬＢ１５２６Ｒ、青色光：ＬＢ１５２６Ｂ、すべてビームテック社製）を用いた。図２に示すように、白色光は４５０ｎｍ及び５８２ｎｍにピークを持つ連続した光波長を有する。緑色光の波長は５１８ｎｍにピークを有する。なお、赤色光の波長は６３３ｎｍにピークを有する。青色光の波長は４６５ｎｍにピークを有する。 A commercially available 31.5 × 18.5 × 24.4 cm water tank of about 12 L was used for breeding medaka. As the light source, an LED (green light: LB1526G, white light: LDA6NG, red light: LB1526R, blue light: LB1526B, all manufactured by Beamtec Co., Ltd.) that emits green light, white light, red light, or blue light was used. . As shown in FIG. 2, white light has a continuous light wavelength with peaks at 450 nm and 582 nm. The wavelength of green light has a peak at 518 nm. The wavelength of red light has a peak at 633 nm. The wavelength of blue light has a peak at 465 nm.
各光源から照射される光の波長のピークのおける放射照度は１Ｗ／ｍ２とした。明暗周期は、明期１４時間及び暗期１０時間とした。明期にのみ光源から光を照射した。すべての光源間で、光強度に有意な差はなかった。水温は約２６℃とした。メダカには、配合飼料（メダカのエサ、キョーリン社製）とアルテミア（ブラインシュリンプエッグス−９０、北村社製）とを１日３回給餌した。 The irradiance at the peak of the wavelength of light emitted from each light source was 1 W / m 2 . The light-dark cycle was 14 hours light and 10 hours dark. Light was emitted from the light source only during the light period. There was no significant difference in light intensity among all light sources. The water temperature was about 26 ° C. The medaka was fed with mixed feed (medaka food, manufactured by Kyorin) and Artemia (brine shrimp eggs-90, manufactured by Kitamura) three times a day.
親メダカから採卵した卵を２６℃のインキュベーターで保温し孵化後２４時間以内の稚魚を各色の試験区に収容した。約３か月飼育し、遺伝的性と表現型の性とを判別した。 Eggs collected from the parent medaka were kept warm in an incubator at 26 ° C., and fry within 24 hours after hatching were housed in each color test area. The animals were raised for about 3 months, and the genetic sex and phenotypic sex were determined.
遺伝的性を示す体色は、色素顆粒によって決まる。色素顆粒は、孵化前より観察できる。本実施例では、より明瞭に判定できる約２か月飼育した個体の体色を、オス及びメスの判定に用いた。遺伝的性がメスの場合、体色は白色である。遺伝的性がオスの場合、体色は緋色である。本実施例では、二次性徴が出現する期間まで飼育した個体を解析に用いた。 The body color indicating genetic sex is determined by pigment granules. Pigment granules can be observed before hatching. In this example, the body color of an individual bred for about two months that can be more clearly determined was used for male and female determination. When the genetic sex is female, the body color is white. When the genetic sex is male, the body color is dark blue. In this example, individuals reared until the period when secondary sexual characteristics appear were used for analysis.
遺伝的性を判別するために、ｄｍｙプライマーを用いたＰＣＲによって性決定遺伝子の有無を決定した。メダカの鰭を５ｍｍほど裁断し、ｍｏｕｓｅ ｔａｉｌ ｂｕｆｆｅｒ（１００ｍＭ Ｔｒｉｓ−Ｃｌ、５ｍＭ ＥＤＴＡ、２００ｍＭ ＮａＣｌ、０．２％ＳＤＳ及び１μＬ Ｐｒｏｔｅｉｎａｓｅ Ｋ）を５００μＬ入れたエッペンチューブ内に鰭を入れ、ブロックインキュベータ上で５５℃に維持し、一晩おいて溶かした。その後、１３０００×ｇで１０分遠心し、上澄みを新しいエッペンチューブに移した。イソプロパノールを５００μＬ入れ、ゲノムＤＮＡを沈殿させ、１３０００×ｇで５分遠心し、ゲノムＤＮＡを回収した。続いて、７０％エタノールで洗浄した後、１０〜２０μＬのＲＮＡａｓｅ ｆｒｅｅ ｗａｔｅｒで希釈した。ｄｍｙプライマーにおけるフォワードプライマー及びリバースプライマーの塩基配列を、それぞれ配列番号１及び配列番号２に示す。 In order to discriminate genetic sex, the presence or absence of a sex-determining gene was determined by PCR using dmy primers. Cut the medaka cocoon about 5 mm, put 500 Ｌ of mouse tail buffer (100 mM Tris-Cl, 5 mM EDTA, 200 mM NaCl, 0.2% SDS and 1 μL Proteinase K) into the Eppendorf tube, and place it on the block incubator. Maintained at 55 ° C. and allowed to dissolve overnight. Thereafter, the mixture was centrifuged at 13000 × g for 10 minutes, and the supernatant was transferred to a new Eppendorf tube. 500 μL of isopropanol was added to precipitate genomic DNA, and centrifuged at 13000 × g for 5 minutes to recover genomic DNA. Subsequently, after washing with 70% ethanol, it was diluted with 10 to 20 μL of RNAase free water. The base sequences of the forward primer and the reverse primer in the dmy primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
ｄｍｙプライマーを用いたＰＣＲによって、遺伝的性がオスの場合、ｄｍｙ遺伝子及びＤｍｒｔ１遺伝子がそれぞれ９００ｂｐ及び１３００ｂｐのバンドとして検出される。遺伝的性がメスの場合、Ｄｍｒｔ１遺伝子のみが１３００ｂｐのバンドとして検出される。ＰＣＲでは、９６℃で予備加熱を行い、９４℃で２分間、９４℃で１０秒、６０℃で３０秒、７２℃で１分を４０サイクル行い、最後に７２℃で７分の伸長反応を行った。酵素としては、ＢＩＯＬＩＮＥ社のＢｉｏｔａｑを使用した。 When genetic sex is male by PCR using the dmy primer, the dmy gene and the Dmrt1 gene are detected as 900 bp and 1300 bp bands, respectively. When the genetic sex is female, only the Dmrt1 gene is detected as a 1300 bp band. In PCR, preheating is performed at 96 ° C, 40 cycles of 94 ° C for 2 minutes, 94 ° C for 10 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally an extension reaction at 72 ° C for 7 minutes. went. Bioline Biotaq from BIOLINE was used as the enzyme.
表現型の性を判別するために生殖腺の組織切片を作成した。メダカから生殖腺を採取し、浸透機の上で一晩、飽和ピクリン酸、ホルマリン及び酢酸を１５：５：１の比率で混合したブアン氏液にて固定した。その後、７０％、８０％、９０％、１００％のエタノールに順次浸け、脱水した。続いて、キシレンに３時間浸けて透徹を行い、パラフィンに包埋した。包埋後にトリミングを行い、ミクロトーム（ＨＭ ３１５Ｓ、ＭＩＣＲＯＭ ｉｎｔｅｒｎａｔｉｏｎａｌ ＧｍｂＨ社製）を用いて４から６μｍの厚さに薄切し、ヘマトキシリン・エオシン染色を行い、観察した。 To determine the phenotypic sex, gonad tissue sections were made. The gonads were collected from the medaka and fixed on a permeator overnight with Buan's solution in which saturated picric acid, formalin and acetic acid were mixed at a ratio of 15: 5: 1. Thereafter, it was immersed in 70%, 80%, 90% and 100% ethanol in order and dehydrated. Subsequently, it was soaked in xylene for 3 hours for penetration and embedded in paraffin. Trimming was performed after embedding, and the slice was sliced to a thickness of 4 to 6 μm using a microtome (HM 315S, manufactured by MICROM International GmbH), followed by hematoxylin / eosin staining and observation.
（結果）
図３及び図４は、それぞれ白色光試験区及び緑色光試験区で飼育したメダカを対象とするｄｍｙプライマーを用いたゲノムＰＣＲの結果を示す。双方の試験区において、体色が白色であるメダカではＤｍｒｔ１遺伝子のバンドのみが検出され、体色が緋色であるメダカではＤｍｒｔ１遺伝子及びｄｍｙ遺伝子の２つのバンドが検出された。よって、緑色光試験区であってもＰＣＲで判別される遺伝的性は、体色が示す遺伝的性と一致した。
(result)
FIG. 3 and FIG. 4 show the results of genomic PCR using dmy primers for medaka fish raised in the white light test group and the green light test group, respectively. In both test groups, only the Dmrt1 gene band was detected in the medaka having a white body color, and two bands of the Dmrt1 gene and the dmy gene were detected in the medaka having a dark body color. Therefore, even in the green light test section, the genetic sex determined by PCR coincided with the genetic sex shown by body color.
図５は、白色光試験区で飼育したメダカの表現型の性を組織観察によって評価した結果を示す。遺伝的性がメスである個体は、すべて卵巣を有していた。一方、遺伝的性がオスである個体は、すべて精巣を有していた。よって、白色光試験区で飼育したメダカでは、遺伝的性が表現型の性と一致し、性転換は誘導されなかった。なお、赤色光及び青色光の試験区でも同様に遺伝的性が表現型の性と一致し、性転換は誘導されなかった。 FIG. 5 shows the result of histological evaluation of the phenotypic sex of medaka bred in the white light test area. All individuals whose genetic sex was female had ovaries. On the other hand, all individuals whose genetic sex was male had testes. Therefore, in the medaka bred in the white light test plot, the genetic sex was consistent with the phenotypic sex, and no sex change was induced. In the red light and blue light test sections, the genetic sex was also consistent with the phenotypic sex, and no sex change was induced.
図６は、緑色光試験区で飼育したメダカの表現型の性を組織観察によって評価した結果を示す。遺伝的性がメスである個体の２７％が精巣を有していた。一方、遺伝的性がオスである個体は、すべて精巣を有していた。よって、緑色光試験区で飼育したメダカでは、遺伝的性がメスである個体のみの性転換が誘導されることが示された。 FIG. 6 shows the results of evaluating the phenotypic sex of medaka bred in the green light test plot by histological observation. Twenty-seven percent of individuals whose genetic sex is female had testes. On the other hand, all individuals whose genetic sex was male had testes. Therefore, it was shown that in the medaka bred in the green light test area, sex change is induced only in individuals whose genetic sex is female.
（実施例２）
実施例１で得られたメスからオスに性転換された個体（偽オス）の精子を、ヒメダカの卵子に人工受精させることで、当該精子が機能的であるか否かを確認した。人工授精には、人工精漿を用いた。人工精漿は、ＮａＣｌ ６５０ｍｇ、ＫＣｌ ４０ｍｇ、ＣａＣｌ２ １１．３ｍｇ、ＭｇＳＯ４・７Ｈ２Ｏ １５ｍｇに対して、蒸留水を１００ｍＬまでメスアップしたものである。偽オスの腹部から圧出した精子を回収した。また、未受精卵は、人工精漿中にてメスを解剖することで得た。初めにホールスライドグラスに未受精卵を乗せ、人工精漿を卵が空気中に露出しない程度に減らした。そして、精子をピペットマンで採集し、ピペッティングによって未受精卵に吹きかけた。その後、メチレンブルーを少量入れ２６℃のインキュベーターを用いて孵化まで加温した。
(Example 2)
It was confirmed whether or not the spermatozoa were functional by artificially fertilizing the sperm of an individual (pseudo male) obtained by transsexualization from female to male obtained in Example 1 into a medaka egg. Artificial seminal plasma was used for artificial insemination. The artificial seminal plasma is obtained by measuring distilled water up to 100 mL with respect to NaCl 650 mg, KCl 40 mg, CaCl 2 11.3 mg, MgSO 4 .7H 2 O 15 mg. Sperm squeezed out from the false male abdomen was collected. Unfertilized eggs were obtained by dissecting a female in artificial seminal plasma. First, an unfertilized egg was placed on the whole slide glass, and the artificial seminal plasma was reduced so that the egg was not exposed to the air. Then, sperm was collected by a pipetman and sprayed on unfertilized eggs by pipetting. Thereafter, a small amount of methylene blue was added and the mixture was warmed to hatch using a 26 ° C. incubator.
（結果）
図７は、偽オスから回収した精子の外観を示す。精子の形態に異常は認められなかった。図８は、偽オスから回収した精子のゲノムＤＮＡ及び偽オスの鰭から抽出したゲノムＤＮＡを対象とするｄｍｙプライマーを用いたゲノムＰＣＲの結果を示す。精子及び鰭のゲノムＤＮＡには、Ｄｍｒｔ１遺伝子のみのバンドが検出され、ｄｍｙ遺伝子のバンドは検出されなかった。
(result)
FIG. 7 shows the appearance of sperm collected from pseudo males. There was no abnormality in the sperm morphology. FIG. 8 shows the results of genomic PCR using dmy primers targeting sperm genomic DNA recovered from pseudomale and genomic DNA extracted from pseudomale sputum. Only the Dmrt1 gene band was detected in the sperm and sputum genomic DNA, and the dmy gene band was not detected.
図９は、Ｆ１世代である受精卵又は孵化した仔魚から抽出したゲノムＤＮＡを対象とするｄｍｙプライマーを用いたゲノムＰＣＲの結果を示す。Ｆ１世代からはｄｍｙ遺伝子のバンドは検出されなかった。よって、Ｆ１個体の遺伝的性はすべてメスであることが示された。 FIG. 9 shows the results of genomic PCR using dmy primers for genomic DNA extracted from fertilized eggs or hatched larvae that are F1 generations. No dmy gene band was detected from the F1 generation. Therefore, it was shown that the genetic sex of F1 individuals is all female.
Ｆ１個体の生殖腺の組織切片を図１０に示す。Ｆ１個体は、卵原細胞と未熟な卵母細胞とを有していた。ゲノムＰＣＲの結果と合わせると、Ｆ１個体は、遺伝的性も表現型の性もメスであった。すなわち、偽オスはＸ染色体を有する精子のみを産生したことが示された。 FIG. 10 shows a tissue section of the gonad of the F1 individual. F1 individuals had oocytes and immature oocytes. Combined with genomic PCR results, F1 individuals were female both genetic and phenotypic. That is, it was shown that the pseudo male produced only sperm having the X chromosome.
上述した実施の形態は、本発明を説明するためのものであり、本発明の範囲を限定するものではない。すなわち、本発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等の発明の意義の範囲内で施される様々な変形が、本発明の範囲内とみなされる。 The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.
本発明は、魚類の養殖に好適である。 The present invention is suitable for fish culture.
１、５ 水槽
２ 光源
３ エアストーン
４ 排水装置
６ ヒーター
７ ポンプ
１００ 性転換誘導装置
DESCRIPTION OF SYMBOLS 1, 5 Aquarium 2 Light source 3 Air stone 4 Drainage device 6 Heater 7 Pump 100 Sex change induction device
Claims (6)
魚類の性転換誘導方法。 Including an irradiation step of irradiating the fish after hatching with green light until sex is determined,
A method for inducing sex change in fish.
５１３〜５２３ｎｍに波長のピークを有する、
請求項１に記載の魚類の性転換誘導方法。 The green light is
Having a wavelength peak at 513-523 nm,
The method for inducing sex change in fish according to claim 1.
性染色体で性が規定される魚類である、
請求項１又は２に記載の魚類の性転換誘導方法。 The fish
A fish whose sex is regulated by sex chromosomes.
The method for inducing sex change in fish according to claim 1 or 2.
性染色体で規定される性がメスである、
請求項３に記載の魚類の性転換誘導方法。 The fish
The sex defined by the sex chromosome is female,
The method for inducing sex change in fish according to claim 3.
魚類の性転換誘導装置。 With a light source that emits green light to fish,
Fish sex change induction device.
前記照射ステップにおいて前記緑色光を照射された、性染色体で規定される性がメスである魚類が有する精子により、該魚類の未受精卵を受精させる受精ステップと、
を含む、魚類の性統御方法。 An irradiation step of irradiating the fish after hatching with green light until sex is determined;
A fertilization step of fertilizing an unfertilized egg of the fish by a sperm of a fish whose sex defined by a sex chromosome is irradiated with the green light in the irradiation step;
Including fish sex control methods.
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| CN112262794A (en) * | 2020-10-24 | 2021-01-26 | 武汉市农业科学院 | Method for sustainable large-scale production of all-female mandarin fish |
| CN114751971A (en) * | 2022-03-31 | 2022-07-15 | 中国科学院海洋研究所 | Paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof |
| CN117502338A (en) * | 2023-12-22 | 2024-02-06 | 中国水产科学研究院黄海水产研究所 | Method for plasma mutagenesis of fertilized eggs of prawns at room temperature under normal pressure |
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| JP2013198430A (en) * | 2012-03-26 | 2013-10-03 | Kitasato Institute | Method for culturing pleuronectiformes fish by irradiation of specific wavelength light |
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| JP2009125012A (en) * | 2007-11-26 | 2009-06-11 | Iwate Prefecture | Method for sex manipulation in fish |
| JP2012065625A (en) * | 2010-09-27 | 2012-04-05 | Kinki Univ | Light wavelength control rearing method for larvae of fishes |
| JP2013198430A (en) * | 2012-03-26 | 2013-10-03 | Kitasato Institute | Method for culturing pleuronectiformes fish by irradiation of specific wavelength light |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112262794A (en) * | 2020-10-24 | 2021-01-26 | 武汉市农业科学院 | Method for sustainable large-scale production of all-female mandarin fish |
| CN114751971A (en) * | 2022-03-31 | 2022-07-15 | 中国科学院海洋研究所 | Paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof |
| CN114751971B (en) * | 2022-03-31 | 2023-10-20 | 中国科学院海洋研究所 | Paralichthys olivaceus male-related Dmrt1 recombinant protein and application thereof |
| CN117502338A (en) * | 2023-12-22 | 2024-02-06 | 中国水产科学研究院黄海水产研究所 | Method for plasma mutagenesis of fertilized eggs of prawns at room temperature under normal pressure |
| CN117502338B (en) * | 2023-12-22 | 2024-05-07 | 中国水产科学研究院黄海水产研究所 | Method for plasma mutagenesis of fertilized eggs of prawns at room temperature under normal pressure |
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