JP6837223B2 - How to reduce the biological population - Google Patents

Description

The present invention relates to a technique for reducing the size of a biological population such as an invasive alien species and thus eradicating it.

In large lakes such as Lake Biwa, the destruction of endemic ecosystems due to the large breeding of piscivorous invasive alien fish such as bluegill and largemouth bass, the endangered rare species, and the catastrophic decline of inland fisheries have become major social problems. Although there has been a certain downward trend due to the huge budget and vigorous extinction efforts of various organizations such as the national government, local governments, fishery cooperatives, and civic activities, resources are still available for major catch target fish species such as honmoroko and shrimp. There are no signs of recovery, and endangered species are still in danger of extinction. Even under the current hard extermination efforts, there is no recovery trend in the stock of native fish, and there is concern that the equilibrium state will be highly stable in the future. Conventional coping methods are 1) invasion control, 2) investigation, capture, collection, physical extermination by poisons, 3) mass release of sterilized individuals in limited species such as insects, and they function effectively. Although some cases have been known, there are many cases that have not been removed. In the problem of alien fish, which is currently a major problem, there is currently no concept or method that enables the eradication or quantitative control of invasive alien species. New drastic measures are needed to restore endemic ecosystems and restore inland fisheries to their pre-invasion state.

Non-Patent Document 1 discloses a method for eradicating a specific pest by releasing a male insect that has been sterilized by radiation treatment. In Japan as well, this method has been applied to the melon flies in the Okinawa archipelago and has been successfully eradicated. However, this method requires a large number of infertile individuals, which exceeds the number of wild populations, and therefore requires a large-scale breeding facility and is expensive. In addition, when eradicating organisms with a short survival cycle (for example, melon flies are about one year), the ratio of infertile individuals to the entire population can be reduced by continuing to release a large number of infertile individuals in a short cycle. It must be maintained above a certain level. In addition, due to the large release of infertile individuals, the total number of individuals will temporarily increase significantly, so if harmful organisms are targeted for extermination, the ecosystem may be destroyed and the damage may increase. .. Furthermore, since the extermination effect depends only on the releasing individual, if the release of the infertile individual is stopped or slowed down, the offspring of the remaining individual will proliferate immediately and the number of individuals will recover in a blink of an eye, so-called rebound risk. Accompanied by.

Non-Patent Document 2 proposes a method for eradicating foreign fish by releasing a combination of sex chromosomes (troy chromosomes) and sexes that do not exist in nature and biasing the proportion of sexes in the population. In particular, the release of a super male (YY male) having two Y chromosomes and a sex-converted individual (YY female) to the female (more effective than the YY male release because YY males are mass-produced in the next generation and later) is mentioned above. It was considered to be more effective than the method of releasing infertile males. However, even with this method, the release of YY males requires a considerable number of released individuals (more than 20% of the population of the wild population for YY males), requires a large breeding facility, and is expensive. .. On the other hand, even the YY female release, which requires a relatively small amount of discharge, requires 8% or more of the population of the wild population, and it is costly to change sex. In addition, even with these methods, the total number of individuals temporarily increases significantly, and in the case of YY female release, the released individual lays eggs, so the eradication time is shorter than YY male release, so the total number during that period There are many problems, such as the number of individuals changing at a high level (Non-Patent Document 3), and when harmful organisms are targeted for extermination, the damage may rather increase. In addition, since the extermination depends on the released individual (YY male release) or only the next two generations (YY female release), if the release is stopped, there is a risk of rebound as in the case of infertile males.

Knipling, E. F. (1955) Possibilities of insect control or eradication through the use of sexual sterile males. J. Econ. Entomol. 48: 459-462. JB Gutierrez and JL Teem (2006) A model describing the effect of sex-reversed YY fish in an established wild population: the use of a Trojan Y chromosome to cause extinction of an introduced exotic species. Journal of Theoretical Biology, 241 (22) : 333-341 Hiroshi Hakoyama, 2008 Foreign Fish Control Technology Development Project Report Mathematical analysis of coexistence conditions, population estimation and extermination methods for foreign fish and native fish 1-10

An object of the present invention is to provide a method for eradicating harmful biological populations such as invasive alien species at low cost, in which an increase in the total population is suppressed and the risk of rebound is suppressed.

As a result of diligent studies, the present inventors have obtained these by using a male individual having a gene that specifically sterilizes a female (for example, a functional deletion mutant of a female-specific maturation gene) as a release individual. I found that the problem could be solved. That is, in an environment where the growth of the population is controlled by physical extermination pressure or the like, if a male individual having a female-specific sterilization gene is continuously released into the wild population in a certain amount or more, as a result of mating, the female The proportion of offspring with a specific sterilization gene increases. Of the offspring population, females homozygous for the female-specific sterilization gene are infertile and cannot leave the next generation. On the other hand, since male offspring individuals having a female-specific sterilization gene are fertile, they will continue to leave the next generation having a female-specific sterilization gene by further mating with females in the wild population. When the sterilization gene is homogenized in females, the sterilization gene is lost from the population due to the inability to leave offspring, but in males, the gene is inherited by offspring due to fertility and is not lost. .. In other words, if sterilization of a female is regarded as synonymous with extermination of an individual, the loss of the gene is minimal compared to the extermination of the individual, and the supply of the gene to the population by release exceeds the loss of this gene. It is possible to continue to increase the occupancy of sterilizing genes in the population. As a result, by continuing to release a relatively small number, the occupancy rate of the infertile gene continues to rise, and as a result of the increase in the proportion of infertile female individuals, the fertility of the population is gradually reduced, and finally this The biological population is reduced and eradicated. By multiplexing the genes to be carried (each gene belongs to a different linkage group = sits on a different chromosome), it is possible to increase the proportion of sterilized females that appear. Furthermore, by converting a male individual carrying this female-specific sterilization gene into a super male (YY male), the eradication power is further enhanced by a synergistic effect with the extermination effect of the release of the YY male. In addition, the extermination effect is not limited to the released individuals, but all individuals that have inherited the genes spread by past releases exert the extermination effect, so once the sterilization gene occupancy rate rises and the fertility is reduced, even if it is released. Even if you stop, the population does not recover immediately, and you can avoid the risk of rebound. Even if the size of the population expands due to unexpected changes in the natural environment when the sterilization gene occupancy rate does not rise sufficiently, the sterilization gene occupancy rate once increased is not affected by the size of the population and changes. Since it does not, unlike the release of sterilized males and YY individuals, the past release is not wasted by this, and it is suitable for business development by releasing small amounts.

The inventors have further studied based on the above findings and have completed the present invention.
That is, the present invention is as follows:

[1] A male individual carrying a female-specific sterilization gene is released into a wild population, and the male individual is mated with a female individual in the wild population to be female-specific in the wild population. A method for reducing the size of the wild population, which comprises producing offspring individuals carrying the sterilization gene.
[2] The method according to [1], wherein the female-specific sterilization gene is a functionally deleted variant of the female-specific maturation gene.
[3] The method according to [1] or [2], wherein the releasing male individual carries two or more female-specific sterilization genes.
[4] The method according to any one of [1] to [3], wherein the male individual to be released is a homozygote of a female-specific infertility gene.
[5] The method according to any one of [1] to [4], wherein the male individual to be released is a super male.
[6] The method according to any one of [1] to [5], wherein the release of a male individual carrying a female-specific sterilization gene is repeated a plurality of times.
[7] The method according to [6], wherein the release of a male individual carrying a female-specific sterilization gene is repeated until there are no fertile female individuals in the wild population.
[8] The method according to any one of [1] to [7], wherein the male individual to be released is labeled so as to be identifiable.
[9] Any of [1] to [8], which includes capturing a part of the wild population and, if the captured individual is a male individual released, further releasing it into the wild population. Or the method described.
[10] The method according to any one of [1] to [9], wherein the species of the wild population is a fish.
[11] The method according to any one of [1] to [10], wherein the wild population is an alien species.
[12] A method for producing a progeny individual that homozygically retains a female-specific sterilization gene, which comprises the following steps:
(I) Reproductive cells or primordial germ cells that homozygously retain the female-specific infertility gene are transplanted into the host infertile female individual so as to be incorporated into the germ gland of the host infertile female individual. To lay eggs carrying the female-specific sterilization gene from proto-cells or primordial germ cells,
(II) By mating the egg with a sperm carrying the female-specific sterilization gene, a progeny individual carrying the female-specific sterilization gene homozygously is produced.
[13] The production method according to [12], wherein the female-specific sterilization gene is a functionally deleted mutant of the female-specific maturation gene.
[14] The production method according to [12] or [13], wherein the sperm carrying the female-specific sterilization gene is the sperm of a male individual carrying the female-specific sterilization gene homozygously.
[15] Germ cells or primordial germ cells that homozygically retain the female-specific sterilization gene are derived from supermale individuals that homozygously retain the female-specific sterilization gene. The production method according to any one of [12] to [14], which is a cell.
[16] The production method according to [15], wherein the sperm carrying the female-specific sterilization gene is a sperm of a super-male individual carrying the female-specific sterilization gene homozygously.
[17] The production method according to any one of [12] to [16], wherein the host infertile female individual is an infertile triploid, an infertile hybrid diploid, or an infertile female heterotriploid.
[18] The production method according to any one of [12] to [17], wherein the host infertile female individual is an individual produced by total female production.
[19] The production method according to any one of [12] to [18], wherein the offspring are male.
[20] The production method according to [19], wherein the offspring are supermale.
[21] The method according to [1], which releases a male individual homozygously carrying a female-specific sterilization gene produced by the method according to [21] [19] or [20].

In the present invention, a male individual having a female-specific sterilization gene is released into a wild population, and by mating with a female individual in the wild group, female offspring individuals having a female-specific sterilization gene are produced. While becoming infertile, males remain fertile, resulting in additional next-generation offspring carrying the female-specific sterilization gene. Since the female-specific sterilization gene is infiltrated into the wild population through natural mating, it is possible to eradicate alien species and the like with a relatively small number of released individuals. As a result, the cost of producing the released individual can be suppressed. In addition, since the increase in the total number of individuals due to release can be suppressed to a minimum, the impact on the ecosystem is small and it can be applied to pests. In addition, it is easy to respond to a rapid increase in fertility due to environmental changes and re-invasion after eradication, and the extermination progresses, the population density drops extremely, and the physical extermination rate required for further population reduction. The extermination effect can be maintained even when it becomes impossible to maintain. In addition, male offspring with the female-specific sterilization gene are fertile and will continue to retain the next generation with the female-specific sterilization gene, thus sustaining the effects of past releases and avoiding the risk of rebound. ing. In addition, it is possible to enhance the extermination effect by multiplexing and loading female-specific sterilization genes belonging to different linkage groups (= sitting on different chromosomes). Furthermore, by converting a male individual having a female-specific sterilization gene into a super-male (YY male), it can be used in combination with super-male release, especially at the stage where the population density decreases and the physical extermination power decreases. Then, since the relative ratio of the released individuals increases, the effect of YY release is likely to be exerted, a large extermination power can be exerted, and the eradication time can be shortened.

A simulation of bluegill extermination by releasing YY male individuals is shown. A simulation of bluegill extermination by release of a male homozygous with one female-specific sterilization gene is shown. A simulation of bluegill extermination by release of a male homozygous with two female-specific sterilization genes is shown. A simulation of bluegill extermination by releasing a YY male individual carrying two female-specific sterilization genes in a homozygous manner is shown. A simulation of bluegill extermination is shown when a YY male individual, which is a homozygote of a female-specific sterilization gene, is released and the released individual is excluded from the extermination target as much as possible (the extermination rate of the labeled individual is set to half). .. The estimated genomic structure of bluegill foxl2 is shown. The putative genomic structure of bluegill foxl2 , the ORF nucleotide sequence, and the putative amino acid sequence encoded by it are shown. The design of guide RNA (crRNA) for bluegill foxl2 is shown. The results of confirming the introduction of mutations into the bluegill foxl2 gene by gene editing are shown. Cleavage of the PAM sequence upstream of 3-4 residues results in the loss of 6-13 residues upstream and downstream. The results of confirming that the amino acid sequence of the FoxL2 protein was modified by the introduction of a mutation into the bluegill foxl2 gene by gene editing are shown. XY males carrying three female-specific sterilization genes in homozygotes are released, and the transition of relative abundance in a model combined with physical extermination is shown (Ricker-type density effect). XY males carrying three female-specific sterilization genes in homozygotes are released, and the transition of relative abundance in a model combined with physical extermination is shown (Beverton-Holt type density effect). We release YY females carrying three female-specific sterilization genes in a heterozygous manner, and show the transition of relative abundance in a model combined with physical extermination (Ricker-type density effect). We release YY females carrying three female-specific sterilization genes in a heterozygous manner and show the transition of relative abundance in a model combined with physical extermination (Beverton-Holt type density effect). The transition of the relative amount of resources in the model in which YY males are released and physical extermination is also used is shown (licker type density effect). The transition of relative abundance in a model in which YY males are released and physical extermination is also used is shown (Beverton-Holt type density effect). YY males carrying one female-specific sterilization gene in homozygous form are released, and the transition of relative abundance in a model combined with physical extermination is shown (Ricker-type density effect). YY males carrying one female-specific sterilization gene in homozygous form are released, and the transition of relative abundance in a model combined with physical extermination is shown (Beverton-Holt type density effect). YY males carrying three female-specific sterilization genes in homozygotes are released, and the transition of relative abundance in a model combined with physical extermination is shown (Ricker-type density effect). YY males carrying three female-specific sterilization genes in homozygotes are released, and the transition of relative abundance in a model combined with physical extermination is shown (Beverton-Holt type density effect).

The present invention releases a male individual carrying a female-specific sterilization gene into a wild population, mates the male individual with a female individual in the wild population, and causes female-specificity in the wild population. Provided are a method for reducing the size of the wild population and a method for suppressing the growth of the wild population, which comprises producing offspring individuals carrying the target sterilization gene. Suppression of wild population growth includes not only reducing the size of the wild population, but also slowing the rate of increase in the size of the wild population.

The species of an individual to which the method of the present invention can be applied is not particularly limited as long as it is an organism that reproduces sexually, and includes, for example, plants and animals that reproduce sexually. Examples of sexually reproducing plants include, but are not limited to, moss plants, fern plants, and seed plants. Examples of sexually reproducing animals include, but are not limited to, fish, arthropods (insects, crustaceans, arachnids, etc.), amphibians, reptiles, birds, mammals (excluding humans), and the like. .. The species of the sexually reproducing wild population of interest is preferably animals, more preferably fish.

In male and female homologous plants, the female-specific sterilization gene is defined as a female genital-specific sterility gene, and instead of a male individual carrying the female-specific sterilization gene, a female genital-specific sterility gene is used. Retaining individuals are released, pollen carrying the female genital sterility gene spreads, pollutes another individual in the wild population, and carries the female genital sterility gene in the wild population. Progeny individuals are born. Such an aspect is also within the scope of the present invention.

In one aspect, the wild population to which the methods of the invention are applicable is an alien species. An alien species is an organism that was not originally in the area but came in from another area through human activity. The alien species is preferably an invasive alien species. Invasive alien species are alien species that have a significant impact on the natural environment of the region and may threaten biodiversity. Invasive alien species include bluegill, largemouth bass, smallmouth bass, kadayashi, grass carp, rosy bitterling, channel catfish, cyprinid, etc. in Japan; cyprinid fish such as silver carp in the five major lakes; bluegill in Europe and South Korea, etc. However, it is not limited to these. Examples of invasive alien arthropods include, but are not limited to, melon flies, Argentine ants, insects such as the redback spider, arachnids such as the redback spider, and crustaceans such as the crayfish. .. Examples of invasive alien mammals include, but are not limited to, raccoons, nutrias, feral cats, and mongooses in Japan. Examples of invasive alien species of birds include, but are not limited to, Chinese hwamei and red-billed leiothrix in Japan. Examples of invasive alien reptiles include, but are not limited to, snapping turtles and green anoles in Japan. Examples of invasive alien amphibians include, but are not limited to, bullfrogs, cane toads, and the like.

The method of the present invention is applied to a wild population inhabiting a closed system (for example, a pond, a lake, a swamp, a remote island, etc.) in which the inflow of individuals into the system and the outflow of individuals to the outside of the system are restricted. It is effective, but it can also be applied to wild populations that live in open systems.

In the present invention, the female-specific sterilization gene means that when the gene is homozygous or heterozygous, only the female individual is specifically sterilized or the fertility is reduced, and the fertility of the male individual is reduced. Means a gene that does not impair. Examples of the female-specific sterilization gene include functionally deleted mutants of the female-specific maturation gene. As a female-specific maturation gene that meets this purpose, it is expressed on the somatic cell side such as egg support cells, contributes to female reproductive physiology such as induction of egg maturation, and is expressed on the germ cell side such as egg progenitor cells and egg cells. Genes that do not are preferred. Candidate genes include, for example, β-subunit gene of follicular stimulating hormone (FSH), β-subunit gene of luteinizing hormone (LH), follicular stimulating hormone receptor (FSH-R) gene, FoxL2 gene, and egg yolk protein precursor vitellogenin. Examples include, but are not limited to, a gene (Vg), an egg membrane precursor protein choriogenin gene (Chg) and a ZP protein gene known as proteins constituting the egg membrane.

A functional deletion mutant of a female-specific maturation gene is a mutant in which the normal function inherent in the female-specific maturation gene cannot be fully exerted, for example, a mutant that does not express the female-specific maturation gene at all. Alternatively, a mutant whose expression level is reduced to the extent that the normal function inherent in the female-specific maturation gene cannot be exerted, or a mutant in which the function of the female-specific maturation gene product is completely lost, or a female-specific maturation gene Examples thereof include mutants in which the function of the female-specific mature gene product is reduced to the extent that the original normal function cannot be exhibited. In the present invention, a mutant in which the female-specific maturation gene is not expressed at all, or a mutant in which the function of the female-specific sterilization gene product is completely lost is preferably used. Normally, female heterozygotes of functionally deleted mutants of the female-specific mature gene maintain fertility by normal allele function, but female homozygotes of the functionally deleted mutant of the female-specific mature gene. The conjugate is sterile.

Individuals carrying the female-specific sterilization gene can be created, for example, using genome editing techniques. For example, Zinc Finger Nucleases (ZFNs) and Transcription Activator, which are artificial nucleases designed to specifically cleave the genomic sequence of a target gene (eg, female-specific mature gene) in a fertilized egg of the target species. -By injecting Like Effector Nucleases (TALENs) or injecting fertilized eggs with synthetic RNA and Cas-9 nuclease complementary to the genomic sequence of the target gene (eg, female-specific mature gene) (CRISPR / Cas) System), specifically disrupts the genome of the target gene (eg, female-specific maturation gene) in the fertilized egg, and encodes a functionally deleted variant of the target gene (eg, female-specific maturation gene) on the genome. Get a fertilized egg. By hatching the obtained fertilized egg, an individual encoding a functionally deleted mutant of a target gene (eg, female-specific maturation gene) on the genome can be obtained. When the obtained fertilized egg is a heterozygote of a functionally deleted mutant of a target gene (eg, female-specific mature gene), a homozygous individual is obtained by mating the heterozygotes with each other. You can get it. Individuals with the functional deletion mutations created can be progeny unless they are homozygous in females, and homozygous by mating heterozygous females with heterozygous or homozygous males in the progeny. Male individuals can be obtained. By using this genome editing technology, it is also possible to directly obtain a homozygous individual in which the target gene is disrupted or introduced without requiring backcrossing operation. In addition, genome editing technology can be applied to a wide range of species including fish, mammals, insects, and plants (Joung and Sander, Nat Rev Mol Cell Biol, 2012; Barrangou, Nat Biotechnol, 2012). Importantly, deletion mutations in genes obtained by such genome editing techniques do not correspond to gene recombination and are allowed to be released into the environment. Technically, a functional deletion mutant of a similar gene can be obtained by knocking in the reporter gene, but in this case, unlike the deletion mutation by genome editing, a gene in which a foreign gene is inserted into the genome is obtained. Since it is a mutant, its release into the environment is generally regulated. In fact, it has been reported that genome editing technology was used to create medaka deficient in FSHR (Endocrinology 155: 3136-3145, 2014).

In the present invention, a male individual carrying two or more female-specific sterilization genes located on different chromosomes is preferably used. Even if they are located on the same chromosome, if they are located far on the genetic map and have a distance that allows easy recombination, multiplexing is meaningful. The significance of multiple loading of multiple female-specific sterilization genes is that when released into a wild population, the extermination effect is greater than when a male individual carrying only one female-specific sterilization gene is used. Is increasing. When mating a heterozygous female with a homozygous male, only one female-specific sterilization gene can sterilize only 50% of offspring females, but two genes 75% and three genes 87.5%. , 4 genes can sterilize 93.75%. Male individuals carrying two or more female-specific sterilization genes can also be produced by mating individuals carrying their respective female-specific sterilization genes. For example, in the above genome editing technique, fertilization is performed. Introduce ZFNs or TALENs designed to specifically cleave the genomic sequences of two or more target genes (eg, female-specific mature genes) into eggs, or introduce two or more target genes into fertilized eggs. It can also be produced directly by introducing synthetic RNAs and Cas-9 nucleases that are complementary to the genome sequence of (eg, female-specific mature gene), respectively. Alternatively, by genome editing technology, an individual carrying one type of female-specific sterilization gene is first obtained, and a fertilized egg carrying one type of female-specific sterilization gene is created from the individual, and the fertilized egg is used. In addition, by applying genome editing technology and specifically disrupting the genome of another target gene (eg, female-specific maturation gene), a male individual carrying two or more female-specific sterilization genes can be obtained. You can get it. The larger the number of female-specific infertility genes to be introduced, the better from the viewpoint of enhancing the infertility for the purpose of eradication. For example, male individuals carrying two or more, three or more, and four or more female-specific sterilization genes are used. In order to increase sterilization power for the purpose of continuing to reduce the number of individuals even when the physical extermination pressure when the population density drops extremely decreases, fertility is assumed when not used in combination with YY. Infertility is low when one or two female-specific sterilization genes are loaded for a vigorous target species, and it is necessary to load as many as possible, preferably four or more species are assumed. Theoretically, the upper limit of the number of female-specific sterilization genes to be introduced is not particularly limited, and the larger the number is, the better from the viewpoint of enhancing sterilization power for the purpose of eradication, but the type of parenchymal chromosome It is affected by the number and even the number of candidate genes. Usually, there are 10 or less species and 5 or less species.

The number of female-specific sterilization genes carried in the released male individual is not particularly limited as long as it allows the reduction of the size of the wild population as a result of mating with the female individual in the wild population, but the proliferative power In the case of vigorous and highly invasive fish, loading only one female-specific sterilization gene is expected to reduce the growth speed of the wild population to some extent, but it is difficult to eradicate it. This is because even assuming the best pairing of heterozygous females and homozygous males, one gene can sterilize only 50% of female offspring. In the case of bluegill, it has been experimentally confirmed that it is difficult to suppress growth even when the physical parent fish extermination rate is 50%. Assuming the case of spawning fish species, the sterilization rate of the parent fish is less than 50%, so if the number of individuals decreases and the physical extermination pressure used in combination decreases, this This is because it is expected that the total extermination pressure of the ayu will decrease, and the growth of the individual will become uncontrollable and the state will shift to the equilibrium state. When two female-specific sterilization genes are used, 75% of female offspring of females can be sterilized, assuming pairing of heterozygous females and homozygous males. When converted to the extermination rate of parent fish, it is expected to be about 60%, which is the level at which the population size can be reduced or not. However, in the final phase when the population has decreased extremely, physical extermination pressure cannot be expected at all, and considering the possibility that the proliferative power will increase due to the cancellation of the density effect that was applied, it will actually be in equilibrium at a low level and eradicated. It may not be possible. Therefore, although it depends on the reproductive rate of the target species, when reducing the population size for fish in general, at least 3 or more females are peculiar unless used in combination with a method of reducing females by mounting on super-male individuals. It is preferable to carry the target sterilization gene, and more preferably, four or more female-specific sterilization genes are loaded.

In the method of the present invention, the male individual released into the wild population may be homozygous or heterozygous for the female-specific sterilization gene, but is preferably homozygous. is there. In the case of heterozygous release, the female-specific sterilization gene occupancy rate is 50% at the highest even if the release is continued, and since it does not increase any more, the appearance rate of females that homogenize and become infertile is low and high. No effect can be expected. However, depending on the type of gene, although it does not reach infertility, heterozygotes may have a negative effect on fertility, and in that case, heterozygotes can also be expected to be effective. On the other hand, when considering the release of homozygotes, if the release is continued, the sterilization gene occupancy rate will gradually approach 100%, so the appearance rate of females who become homozygous and infertile is high, and a high effect can be expected. When a male individual carries two or more female-specific sterilization genes, it is preferably homozygous for at least one female-specific sterilization gene, and more preferably all female-specific sterilization genes. Is homozygous for.

In a preferred embodiment, the male individual released into the wild population in the methods of the invention is a supermale. A super male means a male individual (ie, a YY male) having only two Y chromosomes as sex chromosomes. The use of super-male individuals carrying the female-specific sterilization gene is more common than the use of normal male individuals carrying the female-specific sterilization gene (ie, XY males). By adding the effect of increasing the ratio, it can be expected that the extermination effect will be enhanced. For example, in the case of fish, a super-male YY individual administers a female hormone (estrogen) to a male larva (XY) to obtain a femaleized XY individual, and lays eggs on this XY female to produce a Y egg. It can be produced by fertilization of this with Y sperm, which is usually produced by males. In addition, Y eggs are obtained by using a fetal belly technique in which normal male primordial germ cells and spermatogonia are transplanted into infertile female fry by triploidization, etc., and this and Y sperm normally produced by males are combined. It can also be produced by fertilization. In addition, if YY males are produced, all Y eggs can be produced by borrowing these spermatogonia and transplanting them to the abdominal parent, and by mating this with all Y sperms produced by YY males, all YY males can be produced. Can be produced. YY males carrying the female-specific sterilization gene can be created by applying these super-male YY individual creation methods to individuals carrying the female-specific sterilization gene created by the above-mentioned genome editing. .. YY males carrying the female-specific sterilization gene produced can be screened by genetic testing and progeny testing.

Male individuals (preferably super-male individuals) that homozygically retain the female-specific sterilization gene for release can be technically obtained by selecting individuals obtained by mating if they are animals. However, if it is an animal species that requires mass release, such as fish, it is not practical due to labor and cost. From the viewpoint of practical use, in order to omit the sorting work, only male individuals (preferably super-male individuals) that homozygously retain the female-specific sterilization gene are produced (that is, all-homo male production or all-homo). Super-male production) is required, which can be achieved by using abdominal borrowing techniques. On the other hand, in the case of mammals whose fertility is not so high, male individuals (preferably) that retain the female-specific sterilization gene in a homozygous manner by selection of birth individuals after mating or by pre-implantation genetic diagnosis of artificially fertilized eggs. It is possible to select super-male individuals), and there is not a high need for a fetal belly technique that has not yet been developed. Moreover, if it is a plant, it can be grown in large quantities by using callus culture. In the abdominal borrowing technique assuming fish, etc., as a host infertile female individual, an infertile triploid female of a fish of the same species as the transferred germ cell, an infertile hybrid diploid female or an infertile heterogeneous triad due to cross-breeding of a closely related species. Use diploid females. Regarding infertile allotriploids, even if it is an AAB type allotriploid composed of two homologous genomes and one heterologous genome, an ABC type completely allotriploid composed of all three different genomes ( It may be triploid).
The infertile triploids of fish of the same species as the transferred germ cells give a set of chromosomes that are discarded as the second polar body in the second mature division by giving physical stimuli such as high-pressure treatment and temperature treatment to the fertilized egg. It can be created by keeping it inside the cell (blocking the release of the second polar body), but avoiding the possibility of meiosis of the same type of chromosome and laying aneuploid eggs, or the side of the germ cell to be transplanted. A completely aneuploid triploid (triangular aneuploid triploid) is more desirable in order to prevent a malfunction of coordination between the hormone and the receptor in the above. Infertile all-female hybrid diploids can be produced by normal cross-breeding using a combination of viable hybrids that are simply infertile due to inhibition of meiosis rather than hormonal abnormalities. In addition, considering the efficiency of this operation, since it is necessary to perform the abdominal borrowing operation among the fry that cannot distinguish between male and female, these borrowing such as infertile triploids, infertile hybrid diploids, and infertile heterotriploids It is desirable that all parent candidates are female. For that purpose, by orally administering a male hormone or a female hormone synthesis inhibitor or an anti-estrogen agent to a female fry, a functionally converted male = pseudomale who does not have a Y chromosome but has the ability to form sperm ( It is desirable to produce (XX male) and use this sperm. In addition to the method using these hybrids and triploids, diploid females lacking a reproductive-related gene expressed only on the germ cell side can be used as a borrowing parent by genome editing technology. For the techniques for creating these borrowers, see, for example, the following documents.
・ Dawley RM, Graham JH, Schultz RJ. 1985. Triploid progeny of pumpkinseed × green sunfish hybrids. J Hered 76: 251-257.
Dawley, RM 1987. Hybridization and polyploidy in a community of three sunfish species (Pices: Centrarchidae). Copeia: 326-335.
・ Production of Amphidiploid Medaka Oryzias 2 latipes sinensis --2 curvinotus by Gynogenesis with Retention of the Second Polar Body J. Kurita et al. 1993 Nippon Suisan Gakkaishi, 59 (2) 323
・ Creation of completely heterologous triploid of medaka Jun Kurita et al. 1992, Journal of Japanese Society of Fisheries Science, Vol. 58, No. 12, 2311 ~ 2314
・ Cytogenetic Studies on Diploid and Triploid Oogenesis in Interspecific Hybrid Fish Between Oryzias latipes and O. curvinotus J. Kurita et al. 1995 The Journal of Experimental Zoology, 273, 234-241
-Andrew R. Westmaas, Polyploidy Induction in Bluegill Sunfish (Lepomis Macrochirus) Using Cold and Pressure Shocks. Michigan State University. Department of Fisheries and Wildlife, 1992

For example, when a male individual carrying a female-specific sterilization gene is propagated, germ cells or primordial germ cells carrying the female-specific sterilization gene (preferably homozygous for the female-specific sterilization gene) are propagated. Germ cells or primordial germ cells that retain zygotivity) are transplanted into the above-mentioned host infertile female individuals. Examples of germogenic cells include spermatogonia, oogonia and the like. Germ cells or primordial germ cells carrying the female-specific sterilization gene (preferably homozygotes of the female-specific sterilization gene) are preferably male individuals carrying the female-specific sterilization gene (preferably). , A male individual that homozygically retains a female-specific sterilization gene) is a germ cell or a primordial germ cell. Spermatogonia derived from a male individual carrying the female-specific sterilization gene can be obtained by isolating the spermatogonia from the male individual by a method well known to those skilled in the art. Oogonium derived from a male individual carrying a female-specific sterilization gene is obtained by administering a female hormone (estrogen) to the larvae of the male individual to obtain a femaleized individual, and a person skilled in the art from this female individual. By isolating oogonia by a method well known to the above, or by isolating oogonia produced by transplanting spermatogonia of a male individual carrying a female-specific sterilization gene into a host infertile female individual. , Can be obtained. Primordial germ cells derived from a male individual carrying the female-specific sterilization gene can be obtained by isolating the primordial germ cells from the early embryonic embryos of the male individual. The transplanted germ cells or primordial germ cells are taken up by the host germ gland, meiotic, and develop into eggs (X or Y) carrying the female-specific sterilization gene. By mating this egg with a sperm (X or Y) carrying the female-specific sterilization gene, a progeny individual carrying the female-specific sterilization gene homozygously is produced. By selecting male individuals as needed, male individuals (XY or YY) that homozygically retain the female-specific sterilization gene can be obtained. Since it is inefficient to select the released individuals, in order to efficiently produce all male (XY) individuals, female egg protozoa that retain the female-specific sterilization gene in a homozygous manner are used as fetal parents. The resulting whole X egg may be fertilized with all Y sperm of a super male YY male that retains the female-specific sterilization gene in a homozygous manner.

When a super-male individual carrying the female-specific sterilization gene is homozygously propagated, the super-male individual carrying the female-specific sterilization gene (preferably, the female-specific sterilization gene is homozygously retained) is retained. Germ cells or primordial germ cells (YY) derived from (super-male individuals) are transplanted into the above-mentioned host infertile female individuals. The transplanted germ cells or primordial germ cells are taken up by the host germ gland and undergo meiosis, resulting in only eggs (Y) carrying the female-specific sterilization gene (ie, total Y eggs). By mating this egg with a sperm carrying a female-specific sterilization gene whose sex chromosome is Y, only progeny supermale individuals (YY) who carry the female-specific sterilization gene in a homozygous manner Occurs. The sperm is preferably a supermale individual sperm that homozygically retains the female-specific sterilization gene.

Alternatively, the female-specific sterilization gene can be obtained by giving a physical stimulus such as high-pressure treatment or temperature treatment to all Y eggs to generate a female individual of the second polar body release-inhibiting type or the first division-inhibiting type. It is possible to obtain a female progeny supermale individual (YY) (a homozygous supermale individual having a female-specific sterilization gene).

All males (XY) that homozygously retain the female-specific sterilization gene described above or all supermales (YY) that homozygously retain the female-specific sterilization gene. (YY) is required, but there is a problem with the survivability of the YY male itself, and it is possible that it cannot be used. In that case, production of all XY males is required for release, but the following method is possible to produce all XY males without using YY males.

Even if there is a problem with survival, YY embryos are expected to develop at the hatching stage. Therefore, pseudo-female XY eggs that have undergone sex conversion, or eggs laid by borrowed females that have undergone male sperm cell transplantation (mixture of X-egg and Y-egg) and normal XY male sperm (mixture of X-sperm and Y-sperm) ), The primordial germ cells are borrowed from the hatched fry or embryo of the fertilized egg (which is thought to include YY individuals) and transplanted to the abdominal parent. If the transplanted protozoan cells are YY cells, this borrowing parent lays all Y eggs, so by mating this with all X sperms produced by pseudo males (XX males), all males (XY males) are produced. Is possible. Whether or not the germ cells possessed by the borrowing parent are YY can be confirmed by a progeny test.

It should be noted that the release of males that retain the female-specific infertility gene in a homozygous manner is carried out by mixing with XX females that retain the female-specific infertility gene in a homozygous manner, because the female individual is infertile. Since there is no concern about an increase in the number of individuals due to breeding, it is considered that there is no major problem. In that case, mating between a borrowed female parent transplanted with female oval cells that homozygously retain the female-specific sterilization gene and a normal XY male that homozygously retains the female-specific sterilization gene. XX females (infertility) that homozygically retain the resulting female-specific infertility gene and XY males that homozygically retain the female-specific infertility gene may be released without selection. Alternatively, mating a borrowed female parent transplanted with XY male spermocytes that homozygously retains the female-specific sterilization gene with a normal XY male that homozygously retains the female-specific sterilization gene. Then, from the resulting child, XX females (infertility) that homozygically retain the surviving female-specific sterilization gene and XY males that homozygically retain the female-specific sterilization gene are released without selection. You may. YY individuals that occur at the same time die before release if they are not viable, or die after release if they are poorly survived.

The present invention also provides a method for producing a progeny individual (preferably a male individual, more preferably a super-male individual) that homozygically retains the female-specific sterilization gene as described above. The germ cells (eg, spermatogonia, oogonia) and primordial germ cells used for transplantation in the production method of the present invention are collected from male and female individuals and then cryopreserved in an appropriate cryopreservation solution by the time of transplantation. You may keep it.

This abdominal borrowing technique is particularly effective when using a functionally deleted mutant of a female-specific maturation gene as a female-specific sterilization gene. Introducing germ cells can homozygously retain functionally deleted mutants of the female-specific maturation gene, thus failing to express the functional female-specific maturation gene and producing mature eggs themselves. Although it is not possible, if this germ cell is transplanted by the abdominal technique, the support cell of the egg is provided from the host side and expresses a functional female-specific maturation gene, so that a functional deletion mutation of the female-specific maturation gene is performed. This is because it is possible to mature an egg that retains homozygousness.

Only by natural mating between individuals carrying the female-specific sterilization gene, females homozygously carrying the female-specific sterilization gene (functional deletion mutant of the female-specific maturation gene) become infertile. Therefore, it is difficult to procure a large number of male individuals that homozygically carry the female-specific sterilization gene by normal mating. A heterozygous female individual with a functional deletion mutation of a female-specific maturation gene and a male individual carrying a functional deletion mutation with a female-specific maturation gene were crossed, and among the progeny individuals obtained, females were selected. A certain number can be secured by selecting male individuals that retain the functional deletion mutation of the specific mature gene in a homozygous manner, but it requires genotyping for selection and is subject to release. Since it is necessary to breed a large number of heterozygous individuals that cannot be produced, it is wasteful and expensive. In particular, when trying to exterminate specific species in a vast natural ecosystem such as a lake, it is necessary to procure male individuals that homozygically retain a large amount of female-specific sterilization genes. , The cost is enormous and unrealistic. On the other hand, if the above-mentioned abdominal borrowing technique can be used to obtain a small number of male individuals that homozygically retain the female-specific sterilization gene by genome editing technology, female-specific sterilization will be based on this. It is possible to obtain male individuals that homozygically retain the chemical gene in large quantities and at low cost without requiring genotyping, and it is necessary to exterminate alien species in a vast natural ecosystem. It makes it feasible to procure male individuals that homozygically retain a large number of female-specific sterilization genes. In particular, in the production method of the present invention, when a super-male individual having a female-specific sterilization gene homozygously retained as a male individual to supply germ cells or primordial germ cells and sperm is used, a female useful for release is used. Since only supermale individuals that homozygously retain the specific sterilization gene are produced, genotypes are not required and the cost required to maintain individuals that cannot be released can be reduced, resulting in females. A super-male individual that retains a specific sterilization gene in a homozygous manner can be diverted to the production of further offspring individuals. For details on the abdominal borrowing technique, refer to the following documents, for example.
・ T. Okutsu, S. Shikina, M. Kanno, Y. Takeuchi, G. Yoshizaki. 2007. Production of trout offspring from triploid salmon parents. Science 317, 1517.

Male individuals carrying the female-specific sterilization gene obtained as described above are released into the wild population. Male individuals carrying the released female-specific sterilization gene are naturally mated with female individuals in the wild population, and progeny individuals (heterozygotes) carrying the female-specific sterilization gene in the wild population Occurs. Furthermore, a male individual carrying the released female-specific infertility gene is homozygously retained in the female-specific infertility gene by mating with this heterozygotes or by mating between heterozygotes. Progeny individuals are born. Of the offspring population, females that homozygically retain the female-specific sterilization gene are infertile and cannot leave the next generation, so this can be regarded as extermination of spawning female individuals. In exchange for female extermination, the sterilization gene is also lost from the population without being transmitted to offspring. On the other hand, since males carrying the female-specific sterilization gene are fertile, they become female-specific by mating with females in the wild population to become a vector that transmits the female-specific sterilization gene to the next generation. The target sterilization gene continues to survive in the population without loss. It is considered that the extermination effect does not change even if the same number of males and females are exterminated or only half of the females are exterminated. The associated gene loss can be fully covered by the minimum release of males. In other words, as long as the males that carry the female-specific sterilization gene in the homozygous form continue to be released, the female-specific sterilization gene will permeate into the wild population, and the occupancy rate of the sterilization gene will continue to increase. As the proportion of females that can lay eggs decreases and it becomes impossible to leave offspring, the size of the wild population shrinks, and finally, fertile female individuals are excluded from the wild population, and the wild The population is eradicated.

The number of released male individuals carrying the female-specific sterilization gene is not particularly limited as long as it allows the reduction of the wild population cells as a result of mating with female individuals in the wild population. The population of the wild population before the start of release per release is, for example, 1% or more, preferably 5% or more. If the number of individuals released is too small, the gene loss associated with female infertility cannot be covered, and the equilibrium state cannot be reduced any further. Alternatively, the extermination effect is weak, and the period required for the fertile female to disappear from the wild population becomes long. From the viewpoint of enhancing the extermination effect, the larger the number of male individuals carrying the female-specific sterilization gene to be released, the better, and there is no upper limit for the number of individuals, but if the number of released individuals is too large, it will be released. Ecological damage and (in the case of fish) fishery damage caused by individuals occur. Therefore, the number of male individuals carrying the female-specific sterilization gene to be released is, for example, 20% or less, preferably 10% or less of the number of individuals in the wild population. That is, the number of male individuals carrying the female-specific sterilization gene to be released is preferably 1 to 20%, more preferably 5 to 10%, and even more preferably 6 to 9% of the number of individuals in the wild population. %. In addition, since the release is calculated on the assumption that it will be released to the joining group one year after breeding, if it is released in less than one year due to cost etc., the mortality rate during the rest is appropriately taken into consideration. The amount of release is calculated.

The release of the male individual carrying the female-specific sterilization gene may be released only once a year, but may be repeated multiple times as long as the ecosystem is not adversely affected from the viewpoint of enhancing the extermination effect. The interval of release is, for example, 1/3 to 3 times, preferably 1/2 to 2 times, more preferably the same as the spawning cycle of the target species. For example, when targeting a species that lays eggs once a year (eg, fish), for example, once every two months to three years, preferably once every six months to two years, more preferably 1 Once a year, male individuals carrying the female-specific sterilization gene are released. The range of the number of male individuals carrying the female-specific sterilization gene released at each release is based on the above amount divided by the number of releases, but this should be used as long as there is no adverse effect on the ecosystem. It may be exceeded.

From the viewpoint of enhancing the extermination effect, it is preferable that the number of releases is large as long as the ecosystem is not adversely affected. Theoretically, there is no upper limit on the number of releases, but for example, female-specific sterilization genes until it is confirmed that the number of fertile females in the wild population has decreased compared to before the start of release. Repeated release of male individuals that retain. Stopping release at the reduced position should be avoided as it will lead to recovery of the target wild population. Preferably, until the fertile female population in the wild population is no longer confirmed, or until monitoring confirms that the occupancy of the female-specific sterilization gene in the population has reached 100%, or reproduction. The release of male individuals carrying the female-specific sterilization gene is repeated until the development of fry is no longer confirmed during the period. After the presence of fertile females is no longer confirmed, the release of males carrying the female-specific sterilization gene may be stopped, or to reduce the risk of remaining a small number of fertile females. , Male individuals carrying the female-specific sterilization gene for the purpose of suppressing the breeding growth of naturally or artificially transferred individuals from other places, for a certain number of times or until the risk of transfer from other places is completely eliminated. May continue to be released.

In one aspect, in the method of the invention, the release of a male individual carrying a female-specific sterilization gene may be combined with the physical extermination of the wild population (capture of the wild population and exclusion of the system). Good. Alternatively, it is necessary to combine them in the control of the growing population. At this time, if the captured individual is a male individual carrying the released female-specific sterilization gene, further enhancement of the extermination effect can be expected by releasing it again into the wild population. In order to facilitate such re-release, it is preferable that at least the male individual carrying the female-specific sterilization gene to be released is labeled so that it can be identified. Examples of the label of the male individual to be released include, but are not limited to, a fin cut, an illustrator label, and the like. It is preferable to continue the physical extermination of the wild population for as long as possible because a high extermination effect can be expected. For example, until the occupancy of the female-specific sterilization gene in the population reaches a certain percentage by monitoring, for example, 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more. Most preferably, physical extermination of the wild population is continued until the wild population cannot be confirmed by monitoring (occupancy rate 100%). In particular, when a super-male individual is not used as an individual carrying the female-specific sterilization gene (for example, XY male, XY female, YY female), the occupancy rate of the female-specific sterilization gene is 100%. If the physical extermination is stopped before, the population of the population may recover, so until the wild population cannot be confirmed by monitoring (occupancy rate 100%), the physical extermination of the wild population should be carried out. It is desirable to continue. On the other hand, when a super-male individual carrying a female-specific sterilization gene is used, even if the occupancy rate of the female-specific sterilization gene does not reach 100%, it is female-specific in the population. Even if the physical extermination is stopped when the sterilization gene occupancy rate reaches a certain ratio (for example, 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more). , Population populations continue to decline and eradication may be achievable.

The present invention will be described in more detail with reference to the following examples, but the examples are merely examples of the present invention and do not limit the scope of the present invention in any way.

[Test Example 1]
<Simple simulation with fictitious fish species with bluegill in mind>
The conditions of the simulation are as follows.
・ The initial number of individuals is 0 + 10000 fish, 1 + fish 0 + 50% of fish (5000 fish), 2 + fish 1 + 50% of fish (2500 fish), 3 + fish 2 + 50% of fish (1250 fish), 4 + fish. The above is assumed to be 3 + 50% of fish (625 fish), and the mortality rate when moving to the next grade is 50%. Since the base point of the year is just before the breeding season, 1+ fish will be 2 years old. In this model, 2+ fish = (3 year old fish).
・ The density effect is not considered at all, such as the licker type and Beberton Holt type. It was assumed that it would always breed at the assumed maximum value.
-The parent fish extermination rate required to prevent proliferation was estimated to be 60% from the literature of field tests, and the proliferation rate was set so that the population would be in an equilibrium state at the extermination pressure. Specifically, about 16.1 times the females of the age group involved in breeding shift to 0 + male and female resources of the fish in the following year, and the annual growth rate of the entire resource converges to 143.6% unless extermination is performed.
・ Start physical extermination of parent fish with 60% as the initial parent fish extermination rate. From there, we used a model in which the extermination pressure decreases according to the density.
-The parent fish extermination rate was set to the power of 0.6 (initial population / number of individuals in the year).
・ It is a closed system, and there is no import of wild bluegill from outside the system.
-Release 6% of the initial stock to the recruiting group one year after breeding every year.
-Female-specific sterilization genes do not sterilize females in heterozygotes, but sterilize females in homozygotes.
・ Ignore the difference in the abundance ratio between the parent fish of the gene and the difference in the number of eggs laid in each age group, and calculate the ratio of the next generation gene assuming that the ratio of the gene of the whole parent fish and the average of all the parent fish lay eggs. ing.
・ The male-female ratio is 1: 1.
The time when there were no reproductive XX females was defined as the eradication time. In Figures 1 to 5, the vertical axis shows% and the horizontal axis shows the number of years since the start of discharge.

(1) When 6% of the initial resource amount of YY male individuals was released every year, it could not be eradicated even after 100 years (Fig. 1).
(2) When a male individual carrying one female-specific infertility gene homozygously is used, the infertility rate is 50% even in the highest situation, and the sterilization rate of the parent fish required for growth prevention is 60% (infertility). As the extermination rate of parent fish decreased, the amount of resources became equilibrium and eradication was not achieved. The extermination effect was inferior to that when YY male individuals were used (Fig. 2).
(3) When a male individual carrying two female-specific sterilization genes homozygously is used, it can be eradicated (about 75 years until the eradication of a reproductive female individual), and its extermination effect is that of a YY male individual. It was better than when used (Fig. 3).
(4) When a YY male individual carrying two female-specific sterilization genes homozygously was used, a stronger extermination power was obtained due to the effects of both. In the simulation, reproductive female individuals were eradicated about 50 years after the start of release (Fig. 4).
(5) Female-specific sterilization genes A stronger extermination effect was obtained by releasing YY male individuals, which are homozygotes of two genes, and further labeling the released fish and excluding them from the extermination target. Assuming that the extermination rate of released fish was half that of the other, the reproductive female individuals were eradicated in about 30 years from the start of release, and the abundance could be reduced to 23.5% (Fig. 5).

In this simulation, the physical extermination pressure is not 0 because a certain number of individuals caused by the released fish itself is maintained even in the final stage. On the contrary, the presence of released fish keeps the extermination pressure, but assuming the actual situation, most of the released fish are exterminated, which is wasteful in terms of cost. .. Ultimately, we would like to abandon the costly physical extermination, and for that purpose, in order to continue to reduce the number of individuals even if the physical extermination is 0, multiple loading of 4 genes or more that can achieve a high sterilization rate in released fish , Multiple loading on YY individuals, and combinations thereof are desirable.

[Test Example 2]
The relationship between the number of infertility genes carried by the released males and the infertility rate of females of spawning female offspring was investigated. Each female-specific sterilization gene was assumed to be a different linkage group.

The infertility rate was calculated to be 50% for 1 gene, 75% for 2 genes, 87.5% for 3 genes, and 93.75% for 4 genes. Therefore, it was considered that female offspring could be efficiently sterilized by using two or more female-specific sterilization genes.

[Test Example 3]
The number of sterilization genes required to eradicate the population was examined by female-specific sterilization genes. Specifically, the parent fish extermination rate (%) was set stepwise in the range of 36 to 100%, and the number of sterilizing genes required to achieve the parent fish extermination rate on this simulation was examined.

The sterilization rate is higher than the parent fish extermination rate because females reproduce for several years. In other words, continuous 60% extermination of parent fish every year means that 60% of the parent fish will be exterminated in the first year, 60% of the surviving individuals will be exterminated again in the second year, and the same will be exterminated after the third year. This is because it means that it will continue to be done. If it is an annual fish that lays eggs and dies in one year, the parent fish extermination rate and the sterilization rate are the same. Assuming that the parent fish extermination rate required to suppress the increase in population is 60%, this model shows that two or more female-specific sterilization genes are required to suppress population growth. There is. On the other hand, since it is thought that eradication requires continuous extermination of the parent fish at a rate of about 80 to 90%, fish carrying 3 to 5 female-specific sterilization genes are continuously released. Then, even if the density of the parent fish decreases and the physical extermination pressure cannot be guaranteed at all, it is considered possible to eradicate it. If the released fish is YY male, the number of loaded genes required may be small, and if it is not reduced, the eradication time may be shortened.

[Test Example 4]
<Bluegill foxl2 , fshr sequencing, and introduction of mutations into foxl2>
The entire genome sequence of bluegill was determined, and the nucleotide sequences of the female maturation-related genes foxl2 and fshr were searched. The estimated genomic structure of foxl2 is shown in Figures 6 and 7. The estimated cDNA sequence of foxl2 is shown in SEQ ID NO: 1, and the estimated amino acid sequence is shown in SEQ ID NO: 2. The ORF of bluegill foxl2 consisted of 918 bases (including stop codons) and was estimated to be intron-free. The nucleotide sequence of the bluegill foxl2 genome was highly homologous to European sea bass foxl2.

The genome sequence of bluegill fshr was determined as follows. That is, when the cDNA sequence of medaka fshr (NM_001201514) was subjected to blast analysis against the bluegill genome sequence, a part of it hit sca.212 (5Mb). Gene prediction (FGENESH: tonguefish model) in the region around the hit identified a putative gene region encoding a protein of 677 amino acids (or 707 amino acids). When the amino acid sequence having a length of 677 amino acids was submitted to NCBI blastp analysis, it was found that this amino acid sequence had high homology with the amino acid sequence of fish fshr. In particular, the homology of the croaker fshr to the amino acid sequence was 90% or more. Therefore, it was considered that the obtained putative gene region contained bluegill fshr. The fshr gene structure was very similar between medaka and bluegill. In addition, the position and orientation of genes around the fshr gene were conserved between medaka and bluegill.

Based on the acquired bluegill foxl2 and fshr genomic sequences, we designed a specific guide RNA (crRNA) for cleaving and disrupting genes with the CRISPR / Cas9 system. We designed crRNAs in 3 places for foxl2 and 2 places for fshr. The crRNA for foxl2 is shown in Figure 8.

foxl2_g1 (SEQ ID NO: 3) (Fasmac), which is a crRNA against foxl2, is combined with Cas9 (artificial nuclease) mRNA (addgene (pCS2 + hSpCas9) Cas9 RNA or Thermofisher GeneArt® CRISPR nuclease mRNA) and guide RNA. Microinjection into bluegill fertilized eggs together with tracrRNA (Fasmac) resulted in a large number of progeny individuals with mutations introduced into the foxl2 genome. Since most of the amino acid sequence of the FOXL2 protein is deleted by the stop codon generated by the mutation introduction, it was assumed that the function of the FOXL2 protein was severely inhibited in the obtained mutant individual (Fig. 9). And 10).

[Test Example 5]
<Eradication simulation (up to the use of 3 female-specific sterilization genes)>
(Purpose and method)
According to Ferson et al., The effect of "gene suppression method" that keeps releasing individuals with female-specific sterilization genes little by little, gradually increases the occupancy rate of female-specific sterilization genes in the population, and lowers fertility. It was verified by simulation using the reported bluegill population model (Ricker type: R type) and its modified model (Beverton Holt type: BH type) in Lake Hyco in the United States. When the number of female-specific sterilization genes carried on the released males is set to 1, 2 and 3, each female-specific sterilization gene is homozygous for XY males, homozygous for YY males, and YY females. It was verified when it was mounted in a heterozygous manner.

(result)
(1) Gene suppression scenario I: When a female-specific sterilization gene is homozygously loaded on an XY male
When 1 to 3 female-specific sterilization genes were homozygous for XY males, the population could be eradicated in combination with physical extermination. The time required for eradication became shorter as the loading genes were multiplexed with 2 and 3 genes. However, if physical extermination was completely abandoned shortly before eradication, the population recovered and the population could not be eradicated by continued release (Figs. 11 and 123 female-specific sterilization). Shown only when the gene is loaded).

(2) Gene suppression scenario II: When a female-specific sterilization gene is loaded into a YY female in a heterozygous manner
The extermination power was enhanced by the synergistic effect of the YY female and the female-specific sterilization gene. However, by releasing the females that lay eggs, the decrease in the total population was slow, and the total population of about 20% was maintained in both models even when the females were almost eradicated. Females are eradicated quickly, but they do not meet the goal of rapidly reducing total populations to restore the ecosystem. In addition, the population recovered rapidly when the release was stopped immediately before the eradication of females (Fig. 13 and 14 shown only when carrying three female-specific sterilization genes).

(3) Gene suppression scenario III: When a female-specific sterilization gene is homozygously loaded on a YY male
Even in the case of YY male alone, all offspring are male and females are not produced, so when the number of released fish reaches a certain percentage or more, even if physical extermination is abandoned immediately before eradication, the population could be eradicated only by release ( Figures 15 and 16). In addition, when a female-specific sterilization gene is loaded and released into a YY male, the extermination power is enhanced by the synergistic effect of both, and the number of sterilization genes is increased to 1 to 3, so that the eradication time is shortened. (Fig. 17-20).

(Conclusion)
If a female-specific sterilization gene is carried in a YY male, the population can be eradicated only by release even if the extermination is stopped immediately before the eradication (a stage where physical extermination is difficult). At that time, when the female-specific sterilization gene is triple-loaded, the extermination rate is twice that of the YY male, and the extermination effect is as high as that of the YY female. Therefore, it was shown that it is effective to multiplex the female-specific sterilization gene in YY males.

[Test Example 6]
<Eradication simulation (fixed extermination rate)>
The number of years required for eradication when the discharge is changed in the high-survival Beberton-Holt model under the condition of the extermination rate (51.4%) assuming that the population of our plant is in equilibrium at 30% only by physical extermination. Calculated.

* Since there are actually sneakers, it is unlikely that eradication can be achieved under these conditions.
** Assuming that 20% of eggs are fertilized with sneakers.
* Eradication of females is faster in the combination of YY females and YY females and gene suppression than in the combination of YY males and gene suppression, but there is a problem that the total number of individuals does not decrease easily. In terms of total population, the combination of YY males and gene suppression is excellent.

According to the method of the present invention, it is possible to eradicate alien species and the like at low cost while suppressing the impact on the environment as much as possible.

Claims (16)

A male individual carrying the female-specific sterilization gene, which is a functional deletion variant of the female-specific maturation gene, is released into the wild population, and the male individual is mated with the female individual in the wild population. A method of reducing the size of the wild population, which comprises producing progeny individuals carrying the female-specific sterilization gene in the wild population, wherein the species of the wild population is fish and is released. A method in which a male individual carrying 3 or more female-specific sterilization genes . Male individuals release, homozygous female-specific sterilizing gene, claim 1 Symbol placement methods. The method according to claim 1 or 2 , wherein the male individual to be released is a super male. The method according to any one of claims 1 to 3 , wherein the release of a male individual carrying the female-specific sterilization gene is repeated a plurality of times. The method according to claim 4 , wherein the release of the male individual carrying the female-specific sterilization gene is repeated until there is no fertile female individual in the wild population. The method according to any one of claims 1 to 5 , wherein the male individual to be released is labeled so as to be identifiable. The present invention according to any one of claims 1 to 6 , wherein a part of the wild population is captured, and if the captured individual is a male individual released, the release is further included in the wild population. Method. The method according to any one of claims 1 to 7 , wherein the wild population is an alien species. Method for producing offspring individuals homozygously retaining the female-specific sterilization gene, including the following steps:
(I) To incorporate germ cells or primordial germ cells that homozygously retain the female-specific sterilization gene, which is a functional deletion variant of the female-specific maturation gene, into the germ gland of the host infertile female individual. By transplanting into the host infertile female individual, the germ cell or the primordial germ cell can generate an egg carrying the female-specific sterilization gene.
(II) and the egg, by mating the sperm for holding the female-specific sterilizing gene, a female-specific sterilizing gene A to cause offspring individuals held in homozygous,
A method in which the species of an individual is a fish and the offspring are male . The production method according to claim 9 , wherein the sperm carrying the female-specific sterilization gene is the sperm of a male individual carrying the female-specific sterilization gene homozygously. Germ cells or primordial germ cells that homozygously retain the female-specific sterilization gene are germ cells or primordial germ cells derived from supermale individuals that homozygously retain the female-specific sterilization gene. , The manufacturing method according to claim 9 or 10. The production method according to claim 11 , wherein the sperm carrying the female-specific sterilization gene is a sperm of a super-male individual carrying the female-specific sterilization gene homozygously. The production method according to any one of claims 9 to 12 , wherein the host infertile female individual is an infertile triploid, an infertile hybrid diploid, or an infertile female heterotriploid. Host infertility female individual is an individual that has been produced by all the female production, any one Kouki mounting method of manufacturing of claim 9-13. The production method according to any one of claims 9 to 14, wherein the offspring are supermale. The method according to claim 1, wherein a male individual that homozygically retains the female-specific sterilization gene produced by the method according to any one of claims 9 to 15 is released.