Evaluation method, fish production method, and evaluation agent

Abstract

[Problem] Changing the rearing environment of fish from freshwater to saltwater or from saltwater to freshwater requires fish to quickly adapt to the new environment. Therefore, there has been a need for an evaluation method for evaluating in advance whether fish prior to changing the rearing environment have the ability to adapt to the new environment. [Solution] A method for evaluating the saltwater adaptability of fish that includes a step for examining the expression of a gene of the fish, wherein the gene is a control factor of an ion transporter participating in saltwater adaptation and / or a control factor of an ion transporter participating in freshwater adaptation.

Description

Evaluation method, fish production method, and evaluation agent

[0001] The present invention relates to a method for evaluating the seawater adaptability of fish, a method for producing fish including a step of evaluating the seawater adaptability of fish, and an evaluation agent for evaluating the seawater adaptability of fish.

[0002] Aquaculture techniques for efficient fish production have been developed. In particular, in the aquaculture of migratory fish that migrate between rivers and the sea, it may be necessary to change the rearing environment from freshwater to seawater or from seawater to freshwater. In such cases, it is necessary to change the rearing environment without causing stress to the fish. Known techniques developed from this perspective include a smolt induction method that creates a blue atmosphere in a fish rearing tank (see Patent Document 1) and a rearing improvement method that includes a step of adding a polyvalent cation-sensing receptor (PVCR) regulator that increases the expression of PVCR to freshwater (see Patent Document 2).

[0003] Salmonids are euryhaline migratory fish that can adapt to both freshwater and seawater. It is known that the expression of genes involved in osmoregulation in the gills of salmonids changes depending on the surrounding environment. For example, it is known that the expression of Cftr increases in the gills of chum salmon and rainbow trout after they are transferred to seawater (see Non-Patent Documents 1 and 2), and the expression of Slc26a6 increases in the gills of rainbow trout during freshwater acclimation (see Non-Patent Documents 3 and 4).

[0004] JP 2023-173964 A International Publication No. 2002 / 030182

[0005] Gerber L, Jensen FB, Madsen SS (2018) Dynamic changes in nitric oxide synthase expression are involved in seawater acclimation of rainbow trout Oncorhynchus mykiss. Am J Physiol Regul Integr Comp Physiol 314:R552-R562Wong MKS, Nobata S, Hyodo S (2019) Enhanced osmoregulatory ability marks the smoltification period in developing chum salmon (Oncorhynchus keta). Comp Biochem Physiol A Mol Integr Physiol 238:110565Boyle D, Clifford AM, Orr E, Chamot D, Goss GG (2015) Mechanisms of Cl- uptake in rainbow trout: cloning and expression of slc26a6, a prospective Cl- / HCO3- exchanger. Comp Biochem Physiol A Mol Integr Physiol 180:43-50Leguen I, Le Cam A, Montfort J, Peron S, Fautrel A (2015) Transcriptomic analysis of trout gill ionocytes in fresh water and sea water using laser capture microdissection combined with microarray analysis. PloS one 10:e0139938

[0006] When fish are reared in a new environment, such as freshwater or seawater, they must be able to adapt quickly to the new environment. Therefore, a method for evaluating whether fish can adapt to the new environment before the rearing environment is changed was necessary.

[0007] As in Non-Patent Documents 1 to 4, genes that are specifically expressed in seawater or freshwater have been identified in salmonid fish and other fish. However, it was not clear whether the expression of these genes could be used as an indicator for evaluating the adaptability of fish to new environments.

[0008] That is, the present invention is a method for evaluating the seawater adaptability of fish, which comprises a step of examining gene expression in fish, wherein the gene is a regulator of an ion transporter involved in seawater acclimation and / or a regulator of an ion transporter involved in freshwater acclimation.

[0009] Here, the regulatory factor for the ion transporter involved in seawater acclimation may include Ahcyl2, and the regulatory factor for the ion transporter involved in freshwater acclimation may include Ahcyl3. Furthermore, the gene expression in fish may be gene expression in the gills of fish living in freshwater.

[0010] Another present invention is a method for evaluating the seawater acclimation of fish, comprising a step of examining gene expression in fish, wherein the genes are ion transporters and their regulators involved in seawater acclimation and / or ion transporters and their regulators involved in freshwater acclimation.

[0011] Here, the ion transporter involved in seawater acclimation may include Cftr, and the ion transporter involved in freshwater acclimation may include Slc26a6. Furthermore, the gene expression in fish may be gene expression in the gills of fish living in freshwater.

[0012] The steps of these inventions may be steps of quantitatively examining gene expression, or steps of quantifying gene expression. Another aspect of the present invention is a method further comprising a step of comparing the expression levels of an ion transporter or a regulator thereof involved in seawater acclimation with the expression levels of an ion transporter or a regulator thereof involved in freshwater acclimation, or a method further comprising a step of comparing the expression levels of an ion transporter and a regulator thereof involved in seawater acclimation with the expression levels of an ion transporter and a regulator thereof involved in freshwater acclimation.

[0013] In another aspect of the present invention, the method may further include a step of calculating a score indicating the ratio of the expression levels of ion transporters and their control factors involved in seawater acclimation to the expression levels of ion transporters and their control factors involved in freshwater acclimation, and a step of comparing the score with a predetermined threshold value.

[0014] In the present invention, the fish may be a fish belonging to the salmonidae family.

[0015] The method for producing fish of the present invention includes the steps of raising fish in freshwater, evaluating the adaptability of the fish to seawater, and transferring the fish to seawater. In the present invention, the fish may be fish belonging to the Salmonidae family.

[0016] The agent for assessing seawater acclimation of the present invention comprises a reagent for examining gene expression in fish, wherein the gene is a regulatory factor for an ion transporter involved in seawater acclimation and / or a regulatory factor for an ion transporter involved in freshwater acclimation. Here, the regulatory factor for the ion transporter involved in seawater acclimation may include Ahcyl2, and the regulatory factor for the ion transporter involved in freshwater acclimation may include Ahcyl3.

[0017] In another aspect of the present invention, the reagent may be a reagent for quantifying the expression of the gene. Also, in the present invention, the fish may be a fish belonging to the Salmonidae family.

[0018] According to the evaluation method of the present invention, it is possible to accurately determine whether fish have the ability to adapt to a new environment before the environment is actually changed. Furthermore, since the evaluation method of the present invention allows evaluation by collecting only a small amount of gills, it is possible to evaluate the fish without causing their death.

[0019] 1 is a diagram showing the relationship between an index calculated based on gene expression levels in the gills and plasma osmolality in rainbow trout before and 24 hours after seawater transfer. FIG. 2 is a diagram showing changes in an index calculated based on gene expression levels in the gills in rainbow trout before and 24 hours after seawater transfer. FIG. 3 is a diagram showing the relationship between an index calculated based on gene expression levels in the gills and plasma osmolality in rainbow trout before and 24 hours after seawater transfer. FIG. 4 is a diagram showing the relationship between an index calculated based on gene expression levels in the gills and plasma osmolality in rainbow trout before and 24 hours after seawater transfer. Gene expression levels were measured using universal primers. FIG. 5 is a diagram showing the relationship between an index calculated based on gene expression levels in the gills and plasma osmolality in cherry salmon before and 24 hours after seawater transfer. Gene expression levels were measured using universal primers. This figure shows the relationship between plasma osmolality and an index calculated based on gene expression levels in the gills of Atlantic salmon before and 24 hours after seawater transfer. Gene expression levels were measured using universal primers.

[0020] The present invention relates to a method for evaluating the seawater acclimation of fish, which includes a step of examining gene expression in the fish, a method for producing fish, which includes a step of evaluating the seawater acclimation of fish, and an evaluation agent for evaluating the seawater acclimation of fish. The present invention will be described in detail below based on embodiments, but the present invention is not limited to these embodiments.

[0021] In the present invention, seawater may be salt water containing at least chloride ions, but may also contain sodium, calcium, magnesium, potassium, etc. Seawater may be deep ocean water or artificial seawater, but may also include seawater diluted to 70% to 100% by volume, brackish water having a salt concentration above a certain level, and lake water contained in a salt lake.

[0022] The present invention relates to a method for evaluating or predicting the seawater adaptability of fish by examining gene expression in the gills of the fish. Fish whose gene expression is examined are preferably those reared in freshwater. According to the present invention, it is possible to determine in advance whether or not fish reared in freshwater have seawater adaptability or whether or not they have high seawater adaptability before transferring them to seawater.

[0023] Generally, when fish raised in freshwater are transferred to seawater, their plasma osmolality rises within a short period of time. Subsequently, fish that have acclimatized to seawater experience a decrease in plasma osmolality, while fish that are unable to acclimate to seawater experience reduced feeding and other activities, and in the worst case, death. While plasma osmolality varies depending on the fish species and lineage, it is generally maintained at around 300 mOsm / kg during freshwater life. After transfer to seawater, this temporarily rises to 320-500 mOsm / kg, and then, if successful in seawater acclimation, it falls back to the same level as during freshwater life.

[0024] An example of a fish that is acclimated to seawater or has high acclimation to seawater is a fish whose blood osmotic pressure starts to decrease earlier after being transferred to seawater than fish that are not acclimated to seawater or have low acclimation to seawater. Another example of a fish that is acclimated to seawater or has high acclimation to seawater is a fish that has characteristics such as a lower mortality rate after being transferred to seawater and active feeding behavior in a short period of time after being transferred to seawater than fish that are not acclimated to seawater or have low acclimation to seawater.

[0025] In one embodiment, fish with high seawater adaptability refer to fish whose plasma osmolality value at any time after transfer from freshwater to seawater is lower than that of fish with low seawater adaptability.Furthermore, fish with low seawater adaptability refer to fish whose plasma osmolality value at any time after transfer from freshwater to seawater is higher than that of fish with low seawater adaptability.

[0026] Here, the term "any point in time" varies depending on environmental conditions such as the fish species, fish weight, the temperature of the seawater to which they are transferred, the salinity of the seawater to which they are transferred, and the temperature difference between freshwater and seawater, but it can be, for example, 6 to 72 hours or more after the transfer to the seawater, and can be 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 72 hours, or more.

[0027] The fish in the present invention are preferably migratory fish, more preferably sea-run migratory fish, and are, for example, fish belonging to the Salmonidae family, more specifically, rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), cherry salmon (Oncorhynchus masou), Japanese salmon (Oncorhynchus masou ishikawae), sockeye salmon (Oncorhynchus nerka), chum salmon (Oncorhynchus keta), pink salmon (Oncorhynchus gorbuscha), Chinook salmon (Oncorhynchus tshawytscha), Atlantic salmon (Salmo salar), brown trout (Salmo trutta), etc.

[0028] One embodiment of the present invention includes a step of collecting gill filaments from fish reared in freshwater. By collecting only gill filaments from fish, rather than entire gills or gill arches, the possibility of causing death to the fish can be reduced. It is sufficient to collect an amount of gill filaments that allows for gene expression analysis and histological analysis, and it is sufficient to collect one to several gill filaments. It is sufficient to collect the minimum number of gill filaments necessary so that the act of collecting gill filaments does not cause excessive stress to the fish.

[0029] In the present invention, the genes whose expression is examined are one or more selected from the group consisting of ion transporters involved in seawater acclimation, regulatory factors of ion transporters involved in seawater acclimation, ion transporters involved in freshwater acclimation, and regulatory factors of ion transporters involved in freshwater acclimation.

[0030] One embodiment of the present invention includes a step of examining the expression of a regulator of an ion transporter involved in seawater acclimation and / or a regulator of an ion transporter involved in freshwater acclimation. An example of a regulator of an ion transporter involved in seawater acclimation is Ahcyl2, and an example of a regulator of an ion transporter involved in freshwater acclimation is Ahcyl3.

[0031] Ahcyl is an abbreviation for S-adenosylhomocysteine ​​hydrolase-like protein. Ahcyl is known as a regulatory factor with an IRBIT domain. Human Ahcyl is known to regulate the function of various ion transporters by controlling their phosphorylation (see Non-Patent Document 5), but the function of Ahcyl in fish was unknown. The inventors discovered that in salmonid fish, Ahcyl2 enhances the activity of ion transporters involved in seawater acclimation, and Ahcyl3 colocalizes with ion transporters involved in freshwater acclimation and regulates their function.

[0032] [Non-patent Document 5] UniProtKB O43865 SAHH2_HUMAN https: / / www.uniprot.org / uniprotkb / O43865 /

[0033] In one embodiment of the present invention, the genes whose expression is examined include ion transporters and their regulatory factors involved in seawater acclimation, as well as ion transporters and their regulatory factors involved in freshwater acclimation. By examining the expression of ion transporters, as well as the regulatory factors mentioned above, the adaptability of fish can be more accurately evaluated. For example, in rainbow trout, examples of ion transporters involved in seawater acclimation include Cftr, NKAα1b, and NKCC1a, and examples of ion transporters involved in freshwater acclimation include Slc26a6, NKAα1a, NHE3, and NBC. However, because the function of ion transporters may differ depending on the fish species, the present invention is not limited to these specific examples.

[0034] Cftr is an abbreviation for Cystic fibrosis transmembrane conductance regulator. Salmonid fish are known to have two types of Cftr, Cftr1 and Cftr2, both of which are thought to be ion transporters responsible for the excretion of chloride ions in seawater. Compared to Cftr2, Cftr1 expression has been shown to increase more significantly after transfer to seawater, and it has been confirmed to be localized to the apical membrane of chloride cells in seawater. Therefore, it is preferable to examine the expression of at least Cftr1 when examining Cftr expression.

[0035] NKAα1b is Na + , K + NKCC1a is an abbreviation for Na-ATPase α-subunit 1b. + -K + -2Cl - Both are known as ion transporters involved in the excretion of excess salts in the body in seawater.

[0036] Slc26a6 is an abbreviation for Solute Carrier Family 26 Member 6. Slc26a6 is localized in the apical membrane of chloride cells in freshwater and is thought to be an ion transporter responsible for the uptake of chloride ions in freshwater.

[0037] NKAα1a is Na + , K + -ATPase α-subunit 1a, and NHE3 is Na + / H + Exchanger 3 abbreviation, NBC is Na + -bicarbonate cotransporter, and both are known as ion transporters involved in the uptake of salts into the body in freshwater.

[0038] The inventors have demonstrated that Slc26a6 and Cftr1 are localized to the apical membrane of gill chloride cells in salmonid fish during freshwater and seawater adaptation, respectively. They also found that regulatory factors such as Ahcyl2 can colocalize with ion transporters such as Cftr to improve seawater adaptation, and that regulatory factors such as Ahcyl3 can colocalize with ion transporters such as Slc26a6 to improve freshwater adaptation. Furthermore, they found that the adaptability of fish to freshwater or seawater can be assessed by comparing the relative expression levels of regulatory factors such as Ahcyl2 and / or ion transporters such as Cftr with the relative expression levels of regulatory factors such as Ahcyl3 and / or ion transporters such as Slc26a6.

[0039] That is, one embodiment of the present invention is a method for comparing the expression levels of ion transporters and their regulatory factors involved in seawater acclimation with those of ion transporters and their regulatory factors involved in freshwater acclimation. By taking into consideration the expression levels of the regulatory factors for ion transporters as well as the comparison of the expression levels of ion transporters, it is possible to clarify whether fish are ready to acclimate to a new environment.

[0040] That is, in this embodiment, the ratio of the expression levels of ion transporters and their control factors involved in seawater acclimation to the expression levels of ion transporters and their control factors involved in freshwater acclimation is calculated, and the calculated ratio is compared with a predetermined threshold value.If the calculated ratio is greater than the threshold value, it is determined that the adaptation to seawater is high, and if the calculated ratio is less than the threshold value, it is determined that the adaptation to seawater is low.

[0041] Another embodiment of the present invention may be a method comprising a step of comparing the value obtained by multiplying the expression level of an ion transporter involved in seawater acclimation by the expression level of its control factor with the value obtained by multiplying the expression level of an ion transporter involved in freshwater acclimation by the expression level of its control factor.

[0042] Another embodiment of the present invention is a method comprising the step of comparing the expression level of an ion transporter involved in seawater acclimation in the gills of a fish living in freshwater with the expression level of its regulator, and determining if the ratio of the former value to the latter value is relatively high to determine whether the fish has high seawater acclimation, and if the ratio of the former value to the latter value is relatively low to determine whether the fish has low seawater acclimation.

[0043] In another embodiment of the present invention, the method includes a step of calculating a score indicating the ratio of the expression level of an ion transporter and / or a regulator thereof involved in seawater acclimation to the expression level of an ion transporter and / or a regulator thereof involved in freshwater acclimation in the gills of a fish living in freshwater. In another embodiment, the method includes a step of comparing the calculated score with a predetermined threshold, and determining that the seawater acclimation is high if the score is greater than the threshold, and determining that the seawater acclimation is low if the score is less than the threshold.

[0044] In one example of this embodiment, a score is calculated using the expression levels of ion transporters and / or their regulatory factors involved in seawater acclimation as the numerator and the expression levels of ion transporters and / or their regulatory factors involved in freshwater acclimation as the denominator, and the score is compared with a threshold value to evaluate the seawater acclimation of the fish. The score may be calculated by multiplying it by an arbitrary coefficient.

[0045] In another example of this embodiment, a score is calculated using the numerator obtained by multiplying the expression level of an ion transporter involved in seawater acclimation by the expression level of its regulatory factor and the denominator obtained by multiplying the expression level of an ion transporter involved in freshwater acclimation by the expression level of its regulatory factor, and the score is compared with a threshold to evaluate the seawater acclimation of a fish. An arbitrary coefficient may be used to calculate the score.

[0046] An example of the step of determining the threshold is shown below. After individual identification of test fish living in freshwater, a portion of the gill is collected, and the expression levels of the above-mentioned genes are examined to calculate a score. The test fish are transferred to seawater, and blood is collected after the transfer to seawater to obtain plasma osmolality values. The time for blood collection after the transfer to seawater can be adjusted appropriately based on environmental conditions such as the fish species and fish weight, the temperature of the seawater to be transferred, the salinity of the seawater to be transferred, and the temperature difference between freshwater and seawater. For example, the time is 6 to 72 hours after the transfer to seawater, and can be 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, or 72 hours.

[0047] Statistical processing such as regression analysis is performed on the score calculated from gene expression during freshwater life and the plasma osmolality value after transfer to seawater, and the predicted score value when the plasma osmolality becomes constant after transfer to seawater is set as the threshold value.

[0048] The constant value of plasma osmolality after transfer to seawater is, for example, 350 to 320 mOsm / kg, specifically, but not limited to, 350, 345, 340, 335, 330, 325, or 320 mOsm / kg. The constant value can be arbitrarily determined by the user to be an appropriate value based on environmental conditions such as the target fish species and fish weight, the temperature of the seawater to be transferred, the salinity of the seawater to be transferred, and the temperature difference between freshwater and seawater.

[0049] In this embodiment, the score calculated from the gene expression levels during freshwater life is compared with the threshold value set as described above. If the score is a score where the expression levels of ion transporters involved in seawater acclimation, the expression levels of their regulatory factors, and / or a value calculated from these expression levels is used as the numerator and the expression levels of ion transporters involved in freshwater acclimation, the expression levels of their regulatory factors, and / or a value calculated from these expression levels is used as the denominator, if the score is greater than the threshold value, it is predicted that the plasma osmotic pressure after transfer to seawater will be lower than a certain value, and if the score is smaller than the threshold value, it is predicted that the plasma osmotic pressure after transfer to seawater will be higher than a certain value.

[0050] In another embodiment, the score may be a score in which the expression levels of ion transporters involved in freshwater acclimation, the expression levels of their regulatory factors, and / or a value calculated from these expression levels is used as the numerator, and the expression levels of ion transporters involved in seawater acclimation, the expression levels of their regulatory factors, and / or a value calculated from these expression levels is used as the denominator. In such a case, if the score is smaller than a threshold, it is predicted that the plasma osmotic pressure after transfer to seawater will be lower than a certain value, and if the score is greater than the threshold, it is predicted that the plasma osmotic pressure after transfer to seawater will be higher than a certain value.

[0051] In the present invention, the presence of mRNA of the above-mentioned genes may be detected or the mRNA expression may be quantified. However, specific antibodies against the proteins encoded by these genes may also be used to detect the presence of proteins or their intracellular localization, or the amount of protein present or localized may be quantified.

[0052] To quantify the mRNA expression of each gene, the extracted total RNA is reverse transcribed to obtain cDNA, and quantitative PCR is performed using the obtained cDNA as a template. The expression of each gene is quantified relative to the expression level of a housekeeping gene used as an internal standard, but other methods can also be used.

[0053] Primers used in quantitative PCR can be designed appropriately based on the mRNA nucleotide sequence information of each gene. Information on these nucleotide sequences is publicly available in academic databases. For example, in the Reference Sequence Database (RefSeq) provided by NCBI, for rainbow trout, slc26a6 is assigned the accession number KP025965, cftr1 is assigned the accession number XM_036979287.1, cftr2 is assigned the accession number XM_036958645.1, ahcyl2 is assigned the accession number XM_036976868.1, and ahcyl3 is assigned the accession number XM_036947730.1. The nucleotide sequence information can be viewed using these accession numbers.

[0054] In addition, in RefSeq, the base sequence information of cherry salmon slc26a6 can be viewed using the accession numbers XM_064965913.1, cftr1 XM_064951334.1, cftr2 XM_064929362.1, ahcyl2 XM_064952984.1, and ahcyl3 XM_064956257.1 or XM_064956256.1.

[0055] In addition, in RefSeq, slc26a6 of Atlantic salmon is XM_014192131.2, cftr1 is NM_001123533.1, cftr2 is NM_001123534.1, ahcyl2 is XM_045697043.1, XM_014148348.2, XM_045697044.1, XM_014147409.2, XM_014147411.2, XM_014147412. The nucleotide sequence information for ahcyl3 can be viewed using the accession numbers XM_014147410.2, XM_014207773.2, XM_014207775.2, XM_014207776.2, or XM_014207777.2, and for ahcyl3, XM_045692884.1, XM_045692886.1, XM_045692885.1, or XM_045688275.1.

[0056] In addition, in RefSeq, the nucleotide sequence information for coho salmon slc26a6 can be viewed using the accession numbers XM_020471445.2, cftr1 XM_031823123.1, cftr2 XM_031801336.1, ahcyl2 XM_031822900.1, and ahcyl3 XM_031803831.1 or XM_031803833.1. Primers can also be designed for other fish species using known nucleotide sequence information.

[0057] Here, in the PCR method, a universal primer that can be used across multiple fish species may be used. Such a primer can be obtained by using the above-mentioned nucleotide sequence information to design a primer that binds to a region with high homology between species. In particular, multiple species of salmonid fish are farmed, and universal primers that can be used across multiple salmonid species may be used.

[0058] Another aspect of the present invention is a method for producing fish, the method comprising the steps of raising fish in freshwater, assessing the adaptability of the fish to seawater, and transferring the fish to seawater. By assessing the adaptability of the fish to seawater before transferring the fish to seawater, appropriate conditions for transferring the fish to seawater can be selected.

[0059] That is, one embodiment of the present invention includes a freshwater rearing step of rearing fish in freshwater, an evaluation step of evaluating the seawater adaptability of the fish, a determination step of determining seawater acclimation conditions from the evaluation results of the evaluation step, and a step of transferring the fish to seawater under the seawater acclimation conditions determined in the determination step.

[0060] Specifically, seawater acclimation conditions include the time conditions for replacing the breeding water of fish from freshwater to seawater. For example, if fish raised in freshwater are directly transferred to seawater, the replacement time is zero, which can be said to be harsh conditions for fish with low seawater acclimation ability. On the other hand, if the condition is to gradually increase the salinity of the breeding water, the longer it takes to reach the same concentration as seawater, the easier it will be for the fish to acclimate to seawater, but this has the disadvantage of requiring a long work period. By finding appropriate seawater acclimation conditions based on the acclimation ability of the fish from the evaluation results of the evaluation step, the work period can be reduced.

[0061] Another aspect of the present invention is an agent for assessing seawater acclimation, comprising a reagent for examining gene expression of an ion transporter and / or its regulatory factor involved in seawater acclimation in fish gills, and / or an ion transporter and / or its regulatory factor involved in freshwater acclimation. Specifically, the reagent may be a primer set used in quantitative PCR using cDNA as a template, a specific antibody against a protein encoded by the above-mentioned gene, and a secondary antibody against the antibody. Another embodiment of the present invention may be any of these reagents used in assessing seawater acclimation.

[0062] Another aspect of the present invention may be a kit for assessing seawater acclimation, including a reagent for examining gene expression of an ion transporter and / or its regulatory factor involved in seawater acclimation in fish gills, and / or an ion transporter and / or its regulatory factor involved in freshwater acclimation. Specifically, the reagent may be a primer set used in quantitative PCR using cDNA as a template, a specific antibody against a protein encoded by the above-mentioned gene, and a secondary antibody against the antibody. Another embodiment of the present invention may be these reagents used in assessing seawater acclimation of fish.

[0063] The present invention will be described in more detail below based on examples, but the examples do not limit the present invention.

[0064] Example 1: Changes in gene expression levels and plasma osmolality before and after seawater transfer

[0065] Rainbow trout (345.2 ± 44.9 g, mean fish weight ± standard deviation) reared in freshwater were transferred to seawater. Gill tissue was collected from five individuals immediately before and 24 hours after seawater transfer. RNA was extracted, and mRNA expression levels of cftr1, slc26a6, ahcyl2, and ahcyl3 genes were measured by real-time PCR. Expression levels of each gene were calculated as relative expression levels using ef-1α as an internal standard. The base sequences of the primers used to measure gene expression levels are shown in Table 1. Blood was also collected at the same time as the gills were collected, and plasma osmolality was measured.

[0066]

[0067] Additionally, the seawater acclimation index (SAI), which serves as an evaluation index, was calculated using the following formula: In the formula, "cftr1," "ahcyl2," "slc26a6," and "ahcyl3" represent the relative expression levels of each gene.

[0068]

[0069] Table 2 shows the gene expression levels, SAI, and plasma osmolality of each individual before and 24 hours after seawater transfer. Before seawater transfer, the plasma osmolality of rainbow trout acclimated to freshwater was 319 mOsm / kg. Following seawater transfer, plasma osmolality increased to 338 mOsm / kg 24 hours later. Expression levels of cftr1 and ahcyl2 genes increased after seawater transfer, whereas expression levels of slc26a6 and ahcyl3 genes decreased. Meanwhile, SAI significantly increased following seawater transfer, from -1.09 ± 0.73 before seawater transfer to 5.49 ± 1.06 24 hours later.

[0070]

[0071] Looking at the relationship between the plasma osmolality of each individual and the expression level of each gene, in individuals 24 hours after transfer to seawater, the higher the expression levels of cftr1 and ahcyl2, the lower the plasma osmolality (R 2 The values ​​were 0.37 and 0.74, respectively. In contrast, the higher the expression levels of slc26a6 and ahcyl3, the higher the plasma osmolality tended to be (R 2 The correlation coefficients were 0.85 and 0.78, respectively. Among them, the correlation coefficients between SAI and plasma osmolality in individuals 24 hours after seawater transfer were the highest (R 2 (Value = 0.95) The relationship between SAI and plasma osmolality before and 24 hours after seawater transfer is shown in Figure 1.

[0072] Example 2: Changes in gene expression levels and plasma osmolality in the same individual before and after seawater transfer

[0073] Four rainbow trout (approximately 400g) reared in freshwater were individually identified by implanting a Passive Integrated Transponder (PIT) tag and then transferred to seawater. Gill samples were collected immediately before and 24 hours after seawater transfer, and the mRNA expression levels of each gene were measured and SAI calculated using the same method as described above. Blood samples were also collected at the same time as gill collection, and plasma osmolality was measured. The results are shown in Table 3. The change in SAI for each individual before (0h) and after (24h) transfer is shown in Figure 2, and the relationship between SAI and plasma osmolality is shown in Figure 3.

[0074]

[0075] The mean SAI for all four individuals before seawater transfer was -3.23 ± 1.12, whereas it increased to 5.25 ± 2.45 24 hours after seawater transfer. The individual with the highest SAI before seawater transfer (No. 4; SAI before seawater transfer = -1.69) also had the highest SAI after seawater transfer (SAI after seawater transfer = 8.30) and the lowest plasma osmolality after seawater transfer (350 mOsm / kg). The other three individuals had SAI before seawater transfer ranging from -4.25 to -3.14, and plasma osmolality after seawater transfer ranging from 388 to 399 mOsm / kg, both of which were higher than that of No. 4. Individual No. 2, which had the second lowest SAI before seawater transfer (SAI = -3.85), died on day 3 of seawater transfer.

[0076] A high correlation was confirmed between the SAI before seawater transfer and the plasma osmolality after seawater transfer (R 2 (Value = 0.799) These results indicate that by examining the expression of these genes in individuals before the transfer to seawater, it is possible to predict plasma osmolality after the transfer to seawater. In other words, by examining the expression of these genes in individuals living in freshwater, it is possible to predict whether or not these individuals have acquired seawater acclimation.

[0077] Example 3: Evaluation of improvement of seawater acclimation in rainbow trout using salt-containing feed

[0078] It is known that feeding rainbow trout reared in freshwater with a diet containing 10% salt by weight (hereinafter referred to as "salt-containing diet") improves their adaptability when they are subsequently transferred to seawater (see Non-Patent Document 6). Therefore, 16 rainbow trout reared in freshwater were fed the salt-containing diet once daily at a satiating rate for three weeks, and their gills were collected to examine gene expression. The fish weight at the time of gill collection was 596.9 ± 141.0 g.

[0079] [Non-patent Document 6] Perry SF, Rivero-Lopez L, McNeill B, Wilson J (2006) Fooling a freshwater fish: how dietary salt transforms the rainbow trout gill into a seawater gill phenotype. J Exp Biol 209:4591-4596

[0080] The gene expression levels are shown in Table 4. As shown in Example 1, the SAI calculated for rainbow trout reared in freshwater without a salt-containing feed was -1.09 ± 1.63, whereas in this example, an SAI of 10.7 ± 4.0 was obtained despite being reared in freshwater. Therefore, it was demonstrated that the evaluation method of this example can evaluate the improvement of seawater acclimation in rainbow trout by a salt-containing feed.

[0081]

[0082] Example 4: Evaluation using salmon / trout general PCR primer set

[0083] A universal primer set was designed for broad application to salmonids. This primer set was designed to be compatible with at least rainbow trout, masu salmon, and Atlantic salmon. Therefore, we evaluated the seawater adaptability of rainbow trout, masu salmon (O. masou), and Atlantic salmon (S. salar). Five rainbow trout (814.7 ± 133.3 g, mean fish weight ± standard deviation) reared in freshwater, 12 masu salmon (56.2 ± 12.3 g, mean fish weight ± standard deviation) reared in freshwater, and 10 Atlantic salmon (131.4 ± 26.5 g, mean fish weight ± standard deviation) reared in freshwater were individually identified by PIT tags. Rainbow trout were transferred from freshwater at 12.5°C to 100% seawater by volume at 15.0°C. Masu salmon and Atlantic salmon were transferred from freshwater at 12.5°C to 70% seawater by volume at the same temperature.

[0084] Gills were collected immediately before the seawater transfer, and the mRNA expression level of each gene was measured by real-time PCR using the primer set listed in Table 5 to calculate the SAI. Blood samples were collected 24 hours after the seawater transfer, and plasma osmolality was measured. The relative expression level of each gene and plasma osmolality for each individual are shown in Tables 6 to 8, and the relationship between the SAI immediately before the seawater transfer and the plasma osmolality 24 hours after the seawater transfer is shown in Figures 4 to 6.

[0085]

[0086]

[0087]

[0088]

[0089] It was shown that the primer set shown in Table 5 makes it possible to measure the mRNA expression levels of cftr1, slc26a6, ahcyl2, and ahcyl3 in at least rainbow trout, cherry salmon, and Atlantic salmon.

[0090] The coefficient of determination (R) between the SAI before seawater transfer and the plasma osmolality after seawater transfer 2 The SAI values ​​were 0.88 for rainbow trout, 0.64 for masu salmon, and 0.32 for Atlantic salmon, and correlations were observed in all cases. This indicates that SAI can be used to evaluate seawater acclimation in fish species other than rainbow trout as well. Regarding the relative expression levels of individual genes, a positive correlation was observed between slc26a6 and ahcyl3 and plasma osmolality in rainbow trout and masu salmon, and a negative correlation was observed between cftr1 and ahcyl2 and plasma osmolality in Atlantic salmon. A positive correlation was observed between slc26a6 and ahcyl3 and plasma osmolality, and a negative correlation was observed between ahcyl2 and plasma osmolality in Atlantic salmon.

[0091] The present disclosure provides, for example, the following inventions: [1] A method for evaluating the seawater adaptability of fish, comprising the step of examining gene expression in fish, wherein the genes include regulatory factors for ion transporters involved in seawater acclimation and / or regulatory factors for ion transporters involved in freshwater acclimation. [2] A method for evaluating the seawater adaptability of fish, comprising the step of examining gene expression in fish, wherein the genes include regulatory factors for ion transporters involved in seawater acclimation and regulatory factors for ion transporters involved in freshwater acclimation, and the step of comparing the expression levels of the regulatory factors for ion transporters involved in seawater acclimation with the expression levels of the regulatory factors for ion transporters involved in freshwater acclimation. [3] A method for evaluating the seawater adaptability of fish, comprising: a step of examining gene expression in fish, wherein the genes include a regulatory factor for an ion transporter involved in seawater acclimation and a regulatory factor for an ion transporter involved in freshwater acclimation; a step of comparing the expression level of the regulatory factor for the ion transporter involved in seawater acclimation with the expression level of the regulatory factor for the ion transporter involved in freshwater acclimation; and a step of evaluating the fish as having a high seawater adaptability if the expression level of the regulatory factor for the ion transporter involved in seawater acclimation is relatively higher than the expression level of the regulatory factor for the ion transporter involved in freshwater acclimation. [4] A method for evaluating the seawater adaptability of fish, comprising the steps of: examining gene expression in fish, wherein the genes include regulatory factors for ion transporters involved in seawater acclimation and regulatory factors for ion transporters involved in freshwater acclimation; comparing the expression levels of the regulatory factors for ion transporters involved in seawater acclimation with the expression levels of the regulatory factors for ion transporters involved in freshwater acclimation; and evaluating the fish as having high seawater adaptability when the ratio of the expression level of the regulatory factors for ion transporters involved in seawater acclimation to the expression level of the regulatory factors for ion transporters involved in seawater acclimation is high. [5] A method for evaluating the seawater adaptability of fish, comprising the steps of examining gene expression in fish, wherein the genes are ion transporters and regulatory factors involved in seawater acclimation, and ion transporters and regulatory factors involved in freshwater acclimation.[6] A method for evaluating the seawater adaptability of fish, comprising: a step of examining gene expression in fish, wherein the genes include ion transporters and their control factors involved in seawater acclimation, and ion transporters and their control factors involved in freshwater acclimation; and a step of comparing the expression levels of the ion transporters and their control factors involved in seawater acclimation with the expression levels of the ion transporters and their control factors involved in freshwater acclimation. [7] A method for evaluating the seawater adaptability of fish, comprising: a step of examining gene expression in fish, wherein the genes include ion transporters and their regulatory factors involved in seawater acclimation, and ion transporters and their regulatory factors involved in freshwater acclimation; a step of comparing the expression levels of the ion transporters and their regulatory factors involved in seawater acclimation with the regulatory factors of the ion transporters involved in freshwater acclimation and their expression levels; and a step of evaluating the fish as having a high seawater adaptability if the expression levels of the ion transporters and their regulatory factors involved in seawater acclimation are relatively higher than the expression levels of the ion transporters and their regulatory factors involved in freshwater acclimation, and evaluating the fish as having a low seawater adaptability if the expression levels of the ion transporters and their regulatory factors involved in seawater acclimation are relatively lower than the expression levels of the ion transporters and their regulatory factors involved in freshwater acclimation. [8] A method for evaluating the seawater adaptability of fish, comprising: a step of examining gene expression in fish, wherein the genes include ion transporters involved in seawater acclimation and their regulatory factors, and ion transporters involved in freshwater acclimation and their regulatory factors; a step of comparing a value obtained by multiplying the expression level of the ion transporter involved in seawater acclimation by the expression level of its regulatory factor with a value obtained by multiplying the expression level of the ion transporter involved in freshwater acclimation by the expression level of its regulatory factor; and a step of evaluating the seawater adaptability of the fish as being high if the ratio of the value obtained by multiplying the expression level of the ion transporter involved in seawater acclimation by the expression level of its regulatory factor to the value obtained by multiplying the expression level of the ion transporter involved in freshwater acclimation by the expression level of its regulatory factor is relatively high, and evaluating the seawater adaptability of the fish as being low if the ratio of the value obtained by multiplying the expression level of the ion transporter involved in seawater acclimation by the expression level of its regulatory factor to the value obtained by multiplying the expression level of the ion transporter involved in seawater acclimation by the expression level of its regulatory factor is relatively low.[9] A method for evaluating the seawater adaptability of fish, comprising: a step of examining gene expression in fish, wherein the genes include ion transporters and their control factors involved in seawater acclimation, and ion transporters and their control factors involved in freshwater acclimation; a step of calculating a score indicating the ratio of the expression level of ion transporters and their control factors involved in seawater acclimation to the expression level of ion transporters and their control factors involved in freshwater acclimation; a step of comparing the score with a predetermined threshold; and a step of evaluating the seawater adaptability of the fish as high or low by comparing the score with the threshold.

[10] A method for evaluating the seawater adaptability of fish, comprising: a step of examining gene expression in fish, wherein the genes include ion transporters involved in seawater acclimation and their regulatory factors, and ion transporters involved in freshwater acclimation and their regulatory factors; a step of comparing the ratio of the value obtained by multiplying the expression level of an ion transporter involved in seawater acclimation by the expression level of its regulatory factor to the value obtained by multiplying the expression level of an ion transporter involved in freshwater acclimation by the expression level of its regulatory factor, with a predetermined threshold; and a step of evaluating the seawater adaptability of fish as high or low by comparing the value obtained by multiplying the expression levels with the threshold.

[11] The method according to any one of [7] to

[10] , wherein a high level of seawater acclimation of a fish is predicted by predicting that the plasma osmolality of the fish will be lower than a certain value 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, or 72 hours after transfer to seawater, and a low level of seawater acclimation of a fish is predicted by predicting that the plasma osmolality of the fish will be higher than a certain value 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, or 72 hours after transfer to seawater.

[12] The method according to any one of [1] to

[11] , wherein the ion transporter involved in seawater acclimation is CFTRL, the regulator of the ion transporter involved in seawater acclimation is AHCYL2, the ion transporter involved in freshwater acclimation is SLC26A6, and the regulator of the ion transporter involved in freshwater acclimation is AHCYL3.

[13] The method according to any one of [2] to

[12] , wherein the expression level is a relative expression level.

[14] The method according to any one of [1] to

[13] , wherein the gene expression is gene expression in gills during a freshwater life.

[15] The method according to any one of [1] to

[14] , wherein the step of examining the gene expression of fish comprises collecting gills of fish during a freshwater life and examining the gene expression in the collected gills.

[16] A kit for evaluating the seawater acclimation of fish, comprising reagents for examining the gene expression of regulators of ion transporters involved in seawater acclimation and regulators of ion transporters involved in freshwater acclimation.

[17] A kit for evaluating the seawater acclimation of fish, comprising reagents for examining the gene expression of regulators of ion transporters involved in seawater acclimation and regulators of ion transporters involved in freshwater acclimation.

[18] A kit described in any of

[16] to

[17] , wherein the ion transporter involved in seawater acclimation is CFTRL, the regulator of the ion transporter involved in seawater acclimation is AHCYL2, the ion transporter involved in freshwater acclimation is SLC26A6, and the regulator of the ion transporter involved in freshwater acclimation is AHCYL3.

Claims

1. A method for assessing the seawater acclimation of fish, comprising examining gene expression in the fish, wherein the gene is a regulator of an ion transporter involved in seawater acclimation and / or a regulator of an ion transporter involved in freshwater acclimation.

2. The method of claim 1, wherein the regulator of an ion transporter involved in seawater acclimation comprises Ahcyl2, and the regulator of an ion transporter involved in freshwater acclimation comprises Ahcyl3.

3. The method according to claim 2, wherein the gene expression is gene expression in gills during freshwater life.

4. A method for assessing the seawater acclimation of fish, comprising the step of examining gene expression in fish, wherein the genes are ion transporters and their regulatory factors involved in seawater acclimation and / or ion transporters and their regulatory factors involved in freshwater acclimation.

5. The method of claim 4, wherein the ion transporter involved in seawater acclimation comprises Cftr and the ion transporter involved in freshwater acclimation comprises Slc26a6.

6. The method according to claim 5, wherein the gene expression is gene expression in gills during freshwater life.

7. The method according to any one of claims 1 to 6, wherein said step is a step of quantifying gene expression.

8. The method according to any one of claims 4 to 6, further comprising a step of comparing the expression level of an ion transporter or a control factor thereof involved in seawater acclimation with the expression level of an ion transporter or a control factor thereof involved in freshwater acclimation.

9. The method according to any one of claims 4 to 6, further comprising a step of comparing the expression levels of ion transporters and their control factors involved in seawater acclimation with the expression levels of ion transporters and their control factors involved in freshwater acclimation.

10. The method according to any one of claims 4 to 6, further comprising the steps of: calculating a score indicating the ratio of the expression levels of ion transporters and their control factors involved in seawater acclimation to the expression levels of ion transporters and their control factors involved in freshwater acclimation; and comparing the score with a predetermined threshold value.

11. The method of claim 10, wherein the fish belongs to the salmonidae family.

12. A method for producing fish, comprising the steps of: raising fish in freshwater; evaluating the adaptability of the fish to seawater by the method according to any one of claims 1 to 6; and transferring the fish to seawater.

13. The method for producing fish according to claim 12, wherein the fish belong to the salmonidae family.

14. A seawater acclimation evaluation agent comprising a reagent for examining gene expression in fish, wherein the gene is a regulator of an ion transporter involved in seawater acclimation and / or a regulator of an ion transporter involved in freshwater acclimation.

15. The seawater acclimation evaluation agent according to claim 14, wherein the regulator of an ion transporter involved in seawater acclimation comprises Ahcyl2, and the regulator of an ion transporter involved in freshwater acclimation comprises Ahcyl3.

16. The evaluation agent according to claim 14 or 15, wherein the reagent quantifies the gene expression.

17. The evaluation agent according to claim 16, wherein the fish belongs to the salmonidae family.

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