CN118160687A - Application of targeting Rbm24a to control fish fertility - Google Patents

Abstract

The invention belongs to the technical field of genetic propagation, and particularly relates to application of targeting Rbm24a to control fish fertility. In particular, the invention has found through research that Rbm24a is a novel fish germplasm component which plays a role of germplasm organization. The loss of function of the parent protein can lead the original germ cells not to form and show sterile phenotype, but does not affect the development of other tissues and organs, so rbm a is a novel target gene for controlling fish fertility. Furthermore, the invention also develops a strategy for inducing degradation of the parent Rbm24a protein by using auxin treatment, thereby realizing control of fish fertility, and simultaneously, the degradation of Rbm24a can be induced by zGrad to generate sterile offspring. The method is convenient, safe and thorough in control of fish fertility, and therefore has good practical application value.

Description

Application of targeting Rbm24a to control fish fertility

Technical Field

The invention belongs to the technical field of genetic reproduction, and particularly relates to the application of targeting Rbm24a to control fish fertility.

Background Art

The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

Fish is a high-quality food source for humans. In my country, its output accounts for 52.2% of aquaculture output (2022 National Fisheries Economic Statistical Bulletin), far exceeding other aquatic species. Therefore, improving the output and quality of economic fish has very important strategic and economic value. Fish gonadal development sometimes has a negative impact on economic traits. This is because gonadal development is very energy-consuming and competes with nutritional growth, resulting in adverse consequences such as slower growth rate and meat quality degradation. Therefore, breeding sterile fish has broad economic value. In addition, control of fertility is also the key to whether transgenic fish can be allowed to be used in production. This is because transgenic fish with excellent traits may escape the breeding environment and enter the nature, and reproduce in large numbers to cause ecological invasion and destruction. From the perspective of ecological security, the release of sterile fry for production is a perfect solution to avoid species invasion. Therefore, it is very important to establish a convenient, safe and thorough method to control the fertility of fish.

Rbm24a is an RNA binding protein. The inventors found in previous studies that, in addition to being expressed in the heart and skeletal muscle, the gene encoding this protein is also very specifically expressed in the sensory organ placode, especially the lens. Further tissue sections revealed that this gene is very specifically expressed in the cytoplasm of differentiating primary and secondary fiber cells. It was ultimately proven that Rbm24a is a key post-transcriptional regulatory factor that regulates the terminal differentiation and transparency of the lens. It regulates the translation efficiency of lens-specific mRNA by controlling the length of the mRNA poly(A) tail in the cytoplasm through the cytoplasmic polyadenylation mechanism (PNAS, 2020, 117(13):7245). However, there have been no reports on its role in fish fertility.

Summary of the invention

In response to the above technical problems, the present invention provides an application of targeting Rbm24a to control fish fertility. Specifically, the present invention has found through research that Rbm24a is a new fish germ plasm component, which plays the function of a germ plasm organizer. The loss of its maternal protein function will lead to the inability to form primordial germ cells and exhibit an infertile phenotype, but will not affect the development of other tissues and organs. Therefore, rbm24a is a new target gene for controlling fish fertility. Furthermore, the present invention has also developed a strategy for inducing the degradation of maternal Rbm24a protein through auxin treatment, thereby achieving control of fish fertility; at the same time, zGrad can also be used to induce Rbm24a degradation to produce infertile offspring. Based on the above research results, the present invention is completed.

In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

The first aspect of the present invention provides an application of targeting Rbm24a in controlling fish fertility.

Specifically, the present invention found in zebrafish experimental research that Rbm24a protein is a new germ cell component in fish, and knocking out rbm24a leads to germ cell-specific loss and infertility. Therefore, it can be seen that targeting Rbm24a can effectively control fish fertility.

Therefore, the application is specifically manifested as: knocking out the gene rbm24a leads to fish germ cell-specific loss and infertility.

A second aspect of the present invention provides a method for controlling fish fertility, the method comprising inducing degradation of fish maternal Rbm24a protein by treating with plant growth hormone;

Alternatively, the control method may also be to induce Rbm24a degradation with zGrad to produce sterile offspring, in which case induction by auxin treatment is not required.

The third aspect of the present invention provides the application of the above control method in any one or more of the following:

(a) Large-scale production of sterile fish;

(b) Select and breed superior fish strains and control their fertility.

Wherein, in the application (a), the sterile fish comprises sterile fish embryos;

In the application (b), controlling fertility is specifically manifested as controlling fish sterility.

Beneficial technical effects of one or more of the above technical solutions:

The above technical scheme discovered for the first time that Rbm24a is a new germ plasm component. The loss of its maternal protein function will lead to the inability to form primordial germ cells and exhibit an infertility phenotype, but will not affect the development of other tissues and organs. Therefore, rbm24a is a new target gene for controlling fish fertility.

The further developed strategy of inducing degradation of maternal Rbm24a protein by auxin treatment can achieve the control of fish fertility. This scheme has incomparable advantages: first, targeting rbm24a, the germ cells are completely removed, and the effect of complete infertility can be achieved; second, it has no effect on the health of the fish, and the specific removal of germ cells does not affect the development of other tissues and organs. Third, human and animal cells do not respond to auxin, so there is no need to worry about the safety of auxin treatment; fourth, the processing procedure is simple, low cost, short time, and can be used to process thousands of embryos on a large scale; at the same time, the above technical scheme also provides the use of zGrad to induce Rbm24a degradation to produce infertile offspring.

The establishment of the rbm24a-gfp-degron KI strain in economic fish can achieve large-scale production of sterile embryos for direct production, and can also be used as a chassis for breeding operations. The selected excellent strains also have the characteristics of controlling fertility, and their sterile embryos can be used for production without worrying about their impact on ecological safety. Therefore, they have good practical application value.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

Figure 1 shows that Rbm24a is a germ plasm protein component found in the examples of the present invention; a, the exogenous DNA insertion site of gene knock-in fish. b, Rbm24a-GFP maternal protein was observed to be specifically localized in the germ plasm and germ cells in rbm24a-gfp KI embryos. c, at the 4-cell stage, Rbm24a protein co-localized with the germ plasm RNA ddx4, dazl and the germ plasm protein Piwil1. d, at 24-hpf (hours after fertilization), Rbm24a co-localized with the germ plasm Ddx4 in PGCs.

Figure 2 shows that knocking out the maternal product of rbm24a in an embodiment of the present invention leads to specific loss of germ cells and infertility; a, a method for producing rbm24a maternal mutants. b and c, the eggs laid by maternal mutant males and wild-type females cannot divide, while wild-type males and wild-type females mate and lay eggs normally. d and e, compared with the wild-type testes located on both sides of the swim bladder, rbm24a maternal mutant males have no obvious testis structure. f and g, rbm24a maternal mutants only contain gonadal somatic cell structures, which are vacuolated and have no germ cells. h, markers for detecting germ cells at 24hpf all show that germ cells are completely missing in rbm24a maternal mutants. i and j, in rbm24a maternal mutants, the germ plasm protein Ddx4 completely disappears. k and l, in rbm24a maternal mutants, the germ plasm protein Piwil1 completely disappears. m and n, injection of YFP-nanos3'UTR reporter gene into wild type showed obvious PGCs, but no brightly fluorescent PGCs were observed in mutants. o, germ cell marker genes were detected at 6 hpf, and no signals were detected in rbm24a mutants. p, single-cell RNA-seq data showed that in 6-hpf rbm24a maternal mutants, the PGC population disappeared completely, while other cell populations did not change significantly.

FIG. 3 shows the construction of rbm24a-gfp-degron KI (a) and the realization of auxin-induced germ cell deletion in this strain (b) in an example of the present invention.

Figure 4 shows the production of infertile offspring based on zGrad-induced Rbm24a degradation in an embodiment of the present invention; a shows that co-expression of GFP fusion protein and zGrad in zebrafish will trigger ubiquitination degradation of GFP fusion protein, b shows two methods of producing infertile offspring, and c shows that the expression of PGC marker gene nanos3 completely disappears in 6hpf and 24hpf embryos injected with zGrad mRNA.

DETAILED DESCRIPTION

It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and/or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and/or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

In a typical embodiment of the present invention, a method for targeting Rbm24a for controlling fish fertility is provided.

Specifically, the present invention found in zebrafish experimental research that Rbm24a protein is a new germ cell component in fish, and knocking out rbm24a leads to germ cell-specific loss and infertility. Therefore, it can be seen that targeting Rbm24a can effectively control fish fertility.

Therefore, the application is specifically manifested as: knocking out the gene rbm24a leads to fish germ cell-specific loss and infertility.

In another specific embodiment of the present invention, a method for controlling fish fertility is provided, the method comprising inducing degradation of fish maternal Rbm24a protein by treating with plant auxin;

In another specific embodiment of the present invention, the method comprises:

The modified fourth exon of rbm24a is knocked into the third intron of the rbm24a gene, and the green fluorescent protein and the degradation subsequence (degron) are fused to the endogenous Rbm24a protein; at the same time, the E3 ubiquitin ligase gene TIR1 that responds to plant hormones is connected next to the rbm24a gene, and the E3 ubiquitin ligase gene TIR1 is driven by a promoter, so that the TIR1 protein is expressed in large quantities in eggs and early embryos; through passaging, the rbm24a-gfp-degron KI homozygous strain is selected; then the early embryos are treated with plant hormones; wherein the promoter is not specifically limited, as long as it can drive the gene to be expressed in large quantities in oocytes. In a specific embodiment of the present invention, the promoter is zp3b (zpc) promoter.

The above-mentioned homozygous gene knock-in fish rbm24a-gfp-degron KI are viable and fertile, but after their early embryos are treated with auxin, the endogenous Rbm24a (with a degron tag) will be recognized by the maternally expressed TIR1, and specifically degraded after being linked to ubiquitin, thereby causing germ plasm assembly disorders and infertility.

In another specific embodiment of the present invention, the auxin is specifically a auxin analog 1-naphthylacetic acid potassium, the application concentration is 1-5 μM (preferably 3 μM), the treatment time is 1-10 hours, preferably 6 hours; in a specific embodiment of the present invention, 3 μM 1-naphthylacetic acid potassium is applied to the fertilized egg for 6 hours, and the primordial germ cells PGC in the embryo are completely absent.

Furthermore, it is also possible to avoid the step of auxin treatment, thus providing another method for controlling fish fertility, the method specifically comprising: co-expressing zGrad on the basis of rbm24a-gfp KI. The zGrad is an artificial E3 ubiquitin ligase targeting GFP fusion protein, comprising a nano antibody targeting GFP (vhhGFP4) and an F-box domain from the zebrafish F-box and WD repeat domain containing 11b (fbxw11b) gene, and co-expressing GFP fusion protein and zGrad in zebrafish will trigger ubiquitination degradation of GFP fusion protein; therefore, introducing zGrad mRNA into fertilized eggs produced by rbm24a-gfp-degron KI homozygous strains can also effectively deplete Rbm24a protein, resulting in an infertility effect.

Of course, if the purpose is large-scale production, Tg (ef1α: zGrad) transgenic fish can be constructed, and the homozygous male fish of this strain can be mated with the homozygous female fish of rbm24a-gfp-degron KI, and the offspring produced will all be infertile.

In another specific embodiment of the present invention, the application of the above control method in any one or more of the following is provided:

(a) Large-scale production of sterile fish;

(b) Select and breed superior fish strains and control their fertility.

Wherein, in the application (a), the sterile fish comprises sterile fish embryos;

In the application (b), controlling fertility is specifically manifested as controlling fish sterility.

In the present invention, the fish is preferably an economic fish. Of course, the fish can also be a fish used for basic research, such as zebrafish, etc., which is not specifically limited here.

The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. In the following examples, the materials, reagents, carriers, strains, etc. used are all obtained from commercial sources unless otherwise specified.

Example

1. Discovered that Rbm24a is an essential germ plasm component of primordial germ cells

To understand the function of the maternal Rbm24a protein, we first investigated the subcellular distribution of the protein in cells during early developmental stages. Since there is currently no specific antibody against the zebrafish Rbm24a protein, we attempted to use an intronic genome editing strategy to construct a knock-in strain with a C-terminal GFP tag linked to the endogenous rbm24a coding region (Figure 1a). By replacing the endogenous last exon with a GFP-tagged version, we successfully obtained F0 embryos of the rbm24a-GFP knock-in strain (abbreviated as rbm24a-GFP KI) with a GFP pattern that closely matched the expression of rbm24a mRNA. The zygotically expressed Rbm24a-GFP protein in these embryos was clearly expressed in the lens, heart, skeletal muscle, and hair cells, indicating that successful knock-in occurred at the early cleavage stage.

Next, we examined the localization of maternal Rbm24a-GFP in early embryos generated from F1 rbm24a-GFP KI fish and found that the GFP signal was localized in the germ plasm and germ cells (Fig. 1b). At the 1-cell stage, Rbm24a-GFP was localized in the form of tiny dots throughout the cortical layers. They actively migrated during the first few cell divisions and accumulated along the cleavage furrow (Fig. 1b). At the 1-somite stage, maternal Rbm24a-GFP protein appeared specifically in germ cells, which were divided into two groups on the left and right, bounded by the notochord (Fig. 1b). At 24 hours postfertilization (hpf), maternal Rbm24a-GFP was still visible in primordial germ cells (PGCs) that had migrated to the genital ridge (arrows in Fig. 1b). Maternal Rbm24a-GFP was present as large granules around the germ cell nuclei, while in the adjacent myotomes, zygotic Rbm24a-GFP was almost evenly distributed in the cells (Fig. 1b). Rbm24a-GFP colocalized with ddx4, dazl, and Piwil1 in early cleavage-stage embryos (Fig. 1c), and at 24 hpf, it perfectly colocalized with the Ddx4 protein in PGCs (Fig. 1d). Since the homozygous rbm24a-GFP KI line was viable and fertile, GFP tagging did not affect the function of the endogenous Rbm24a protein. Therefore, the localization pattern of Rbm24a-GFP should represent the status of the endogenous protein. These results indicate that the Rbm24a protein is a new germ plasm component in fish.

2. Knockout of rbm24a results in a completely sterile phenotype

Since zygotic mutants of rbm24a exhibit lethal phenotypes such as heart failure, cataracts, and hair cell defects, homozygous mutant female fish and their maternal mutant embryos cannot be obtained by traditional genetic methods. The cardiac defects can be rescued by driving the expression of rbm24-gfp using the cmlc2 promoter, but the resulting fish are still not viable because the larvae show a dysequilibrium phenotype. Therefore, to facilitate maternal mutant screening, we first constructed a knock-in strain by fusion of RFP to the C-terminus of the Rbm24a protein and simultaneously inserted a zpc:cas9 vector downstream of the rbm24a gene. We named this knock-in strain rbm24a-RFP KI ^zpc:cas9 . Similar to the rbm24a-GFP KI strain, the homozygous rbm24a-RFP KI ^zpc:cas9 strain is healthy and fertile. The Rbm24a-RFP signal is present in the correct tissues where the endogenous rbm24a gene is expressed. Maternal Rbm24a-RFP also showed germline cytoplasmic localization. Meanwhile, the zpc:cas9 insert supported sufficient maternal expression of Cas9 protein, as injection of bmp2b sgRNA into embryos generated from rbm24a-RFP KI ^zpc:cas9 females resulted in nearly 100% dorsalization phenotype. The high genome editing efficiency of rbm24a-RFP KI ^zpc:cas9 remained stable in three consecutive generations.

On the other hand, we constructed a transgenic vector that expresses a maternal BFP marker and four highly efficient sgRNAs targeting the rbm24a coding sequence. This sgRNA expression vector was introduced into homozygous rbm24a-RFPKI ^zpc:cas9 embryos via Tol2 transposition, and rbm24a maternal mutants (Mrbm24a) were finally obtained in BFP-positive embryos in the next generation (Figure 2a). Mrbm24a mutants were easily picked out because their Rbm24a-RFP signal disappeared (Figure 2a, lower right inset). Mrbm24a embryos looked normal and survived to adulthood. Maternal and zygotic mutants of rbm24a (MZrbm24a) showed the same lethal phenotype as their zygotic mutants, indicating that the maternal Rbm24a protein cannot compensate for the function of zygotic rbm24a. However, Mrbm24a adult fish showed all-male traits. They had mating behavior but could only produce unfertilized eggs (Figure 2b and c) and were therefore sterile. In wild-type males, testes were found on both sides of the swim bladder. In contrast, rbm24a maternally derived mutant fish had deposited adipose tissue where the testes should be (Figure 2d and e). Sectioning and HE staining analysis showed that sperm and immature male germ cells could not be observed in Mrbm24a fish. Instead, only empty testicular tubular structures remained in Mrbm24a mutant fish, which was very similar to the phenotype of dnd1-deficient fish (Figure 2f and g). Due to the lack of germ cells in Mrbm24a adult fish, we speculated that the formation of primordial germ cells may have been disrupted. To investigate this possibility, we examined the expression of several germ plasm mRNAs in rbm24a maternally derived mutants 24 hours after induction, including nanos3, ddx4, ca15b, tdrd7, and kop. Notably, the PGC-specific expression of these germ cytoplasmic mRNAs disappeared in Mrbm24a embryos after 24 hours (Figure 2h). Consistent with this, protein markers of PGCs, such as Piwil1 and Ddx4, could not be detected in mutants by immunofluorescence (Figure 2i-l). We also injected YFP-nanos3-3'UTR mRNA into wild-type and Mrbm24a embryos, but no YFP-positive PGCs could be detected in the latter (Figure 2m and n). The absence of PGCs in mutants could be observed even at the initial developmental stage after 6 hours (Figure 2o). Single-cell sequencing results also showed that Mrbm24a embryos specifically lost the PGC cell type (Figure 2p). Together, these results indicate the complete disappearance of PGCs in rbm24a mutants, and that this gene can serve as a target gene for manipulating fish fertility.

3. Construction of a zebrafish strain with auxin-induced Rbm24a degradation

In order to make Rbm24a respond to auxin-induced protein degradation (AID), we knocked in a gene at the same position of the third intron of rbm24a, so that the endogenous protein of Rbm24a was marked with GFP and a degradation subsequence (Figure 3a). Through subculture, the rbm24a-gfp-degron KI homozygous strain was selected. The fertilized eggs produced by the rbm24a-gfp-degron KI homozygous strain were treated with 3μM K-NAA (1-naphthaleneacetic acid, potassium salt, auxin analog, dissolved in E3 buffer) for 6 hours, and the untreated and treated embryos of 6hpf and 27hpf were fixed, and nanos3 was used as a probe to detect the presence of PGC. The results showed that PGCs were completely absent in embryos treated with auxin (Figure 3b).

Among them, the sgRNA sequence for gene knock-in in the third intron of the rbm24a gene is: taatacgactcactataGGGCTGCTGTCATGTTGGGTgttttagagctagaa (SEQ ID NO.1)

ZPC:TIR1 KI ^{RBM24A 3rd Intron} gene knock-in plasmid sequence information:

Among them, the black bold part is the sgRNA target region, the blue underlined part is the last exon region of rbm24a; the green part is the EGFP sequence; the red underlined part is the degradation subsequence; the orange part is the rbm24a 3' untranslated region; the gold underlined part is the zpc promoter sequence; is the Kozak sequence, is the FLAG sequence, the purple underlined part is the tir1 sequence, and the blue italic part is the SV40 poly(A) signal sequence.

4. zGrad induces Rbm24a degradation to produce sterile offspring

Another idea to control the degradation of Rbm24a is to co-express zGrad on the basis of rbm24a-gfp KI. zGrad is an artificial E3 ubiquitin ligase targeting GFP fusion protein, which contains a nanobody targeting GFP (vhhGFP4) and an F-box domain from the zebrafish F-box and WD repeat domain containing 11b (fbxw11b) gene. Co-expression of GFP fusion protein and zGrad in zebrafish will trigger ubiquitination and degradation of GFP fusion protein (Figure 4a, eLife, 2019, 10.7554). Therefore, injecting zGrad mRNA (100pg/embryo) into fertilized eggs produced by rbm24a-gfp-degron KI homozygous strains can also effectively deplete Rbm24a protein and produce infertility. For the purpose of large-scale production, Tg(ef1α:zGrad) transgenic fish can be constructed, and homozygous male fish of this strain can be mated with homozygous female fish of rbm24a-gfp-degron KI, and the offspring produced will be infertile (Figure 4b). This method can avoid the auxin treatment step, but it is necessary to maintain two strains. As shown in Figure 4c, the expression of PGC marker gene nanos3 completely disappeared in embryos injected with zGrad mRNA at 6hpf and 24hpf, showing the effectiveness of this strategy.

The sequence information of the pCS2-zGrad plasmid used to express zGrad mRNA is as follows:

in, is the Sp6 promoter sequence, the green part is the F-box sequence, the orange part is the GFP nanobody sequence, the pink part is the SV40 poly(A) signal sequence, the green underlined part is the AmpR sequence, the blue part behind it is the AmpR promoter sequence, and the gold underlined part is the CMV IE94 promoter.

It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. Application of targeting Rbm24a in controlling fish fertility. 2. The use according to claim 1, characterized in that the use is manifested in that knocking out the gene rbm24a leads to specific loss of fish germ cells and infertility. 3. The use according to claim 1 or 2, characterized in that the fish include economic fish and fish used for basic research. 4. A method for controlling fish fertility, characterized in that the control method comprises inducing degradation of fish maternal Rbm24a protein by treating with plant growth hormone; specifically, the control method comprises: The modified fourth exon of rbm24a was knocked into the third intron of the rbm24a gene, and the green fluorescent protein and the degradation subsequence were fused to the endogenous Rbm24a protein; at the same time, the E3 ubiquitin ligase gene TIR1 that responds to plant auxin was connected next to the rbm24a gene, and the E3 ubiquitin ligase gene TIR1 was driven by a promoter, so that the TIR1 protein was expressed in large quantities in eggs and early embryos; through passaging, the rbm24a-gfp-degron KI homozygous strain was selected; and then the early embryos were treated with plant auxin. 5. The control method according to claim 4, characterized in that the sgRNA sequence for performing gene knock-in is shown as SEQ ID NO.1; the knock-in plasmid is shown as SEQ ID NO.2; The plant growth hormone is a plant growth hormone analog 1-naphthylacetic acid potassium, the applied concentration is 1-5 μM, and the treatment time is 1-10 hours. 6. A method for controlling fish fertility, characterized in that the method specifically comprises: introducing zGrad mRNA into fertilized eggs produced by the rbm24a-gfp-degron KI homozygous strain to produce infertile offspring fish; Alternatively, Tg(ef1α:zGrad) transgenic fish are constructed and homozygous males of this strain are mated with homozygous females of rbm24a-gfp-degronKI to produce sterile offspring fish. 7. The control method according to claim 6, characterized in that the plasmid expressing zGrad mRNA is as shown in SEQ ID NO.3. 8. Application of the control method according to any one of claims 4 to 7 in any one or more of the following: (a) Large-scale production of sterile fish; (b) Select and breed superior fish strains and control their fertility. 9. The use according to claim 8, characterized in that in the use (a), the sterile fish comprises sterile fish embryos; In the application (b), controlling fertility is specifically manifested as controlling fish sterility. 10. The use according to claim 8, characterized in that the fish include economic fish and fish used for basic research.