Method for regulating the color of ornamental fish

Abstract

The application discloses a method for regulating body color of ornamental fish and belongs to the technical field of aquaculture. The application finds that, by using thyroid hormone to soak ornamental fish in the fry period and the juvenile fish period, excessive thyroid hormone can significantly reduce the number of red pigment cells and the size of pigment granules of ornamental fish in different periods, and reduce the content of carotenoids; when the synthesis of thyroid hormone is inhibited, the expression of melanin cells and related synthesis pathway genes is promoted, and the carotenoid level is increased. The application first clearly defines the inhibitory effect of thyroid hormone on the development of red pigment cells and the accumulation of carotenoids of gourami, fills the knowledge gap in the field, and provides important guiding information for future regulation of body color and genetic improvement of ornamental fish and other aquatic animals.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquaculture, and particularly relates to a method for regulating the body color of ornamental fish. BACKGROUND

[0002] Thyroid hormones (TH), including 3,5,3'-L-triiodothyronine (T3), have long been considered as key factors in regulating the growth, development and metabolism of organisms. In fish, thyroid hormones are essential for metamorphosis and are believed to inhibit the differentiation and distribution of melanophore cells and promote the differentiation of xanthophore and iridophore cells. However, as red is an important economic trait of aquatic animals, relatively little is known about how thyroid hormones affect the development of red pigment cells, especially the specific mechanisms of their effects on carotenoid metabolism. This lack of knowledge limits our understanding of the changes in fish body color and hinders the possibility of optimizing the selection and breeding of ornamental fish varieties through endocrine regulation. In addition, the lack of understanding of the mechanisms of carotenoid accumulation under the regulation of thyroid hormones also affects the effective application of ornamental fish body color in related aquaculture practices.

[0003] Scatophagus argus belongs to Perciformes, Scatophagidae and Scatophagus, and is commonly known as gold drum. As a famous and valuable marine economic fish in the south, Scatophagus argus is one of the valuable ornamental fish species, and is also favored by consumers due to its delicious taste. Scatophagus argus has red pigment cells in the embryonic stage, and forms bright red patches on the back in the post-embryonic stage, which is an ideal model fish for studying the regulation of thyroid hormones on red pigment cells and carotenoids. SUMMARY

[0004] The present application discloses a new function of thyroid hormones (TH), particularly 3,5,3'-L-triiodothyronine (T3), in regulating the body color pattern of ornamental fish, i.e., its inhibitory effect on the development of red pigment cells and the accumulation of carotenoids. In addition, the present application also relates to methods for controlling or changing the body color of ornamental fish using this discovery.

[0005] The present application provides the application of thyroid hormones in improving fish body color products. The present application first clearly demonstrates the inhibitory effect of thyroid hormones on the development of red pigment cells and the accumulation of carotenoids in Scatophagus argus, filling the knowledge gap in this field and providing important guidance for future research on the regulation and genetic improvement of body color in ornamental fish and other aquatic animals.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application provides a method for regulating the body color of ornamental fish, which is as follows:

[0008] Place the post-fertilization embryo development period of ornamental fish in the water body, add thyroid hormone T3 with a final concentration of 0.05-0.15 μg / mL to the water body, and breed for 24-96 h;

[0009] Place the juvenile period of ornamental fish in the water body, add 0.05-0.15 μg / mL of thyroid hormone T3 to the water body, and breed for 20-40 days.

[0010] Further, the pH value of the breeding water body is 6.4±0.2 mg / L.

[0011] Further, the breeding temperature is 25-30℃, and the breeding salinity is 20-30‰.

[0012] Further, the breeding density of the embryo development period during the breeding process is 700-1300 tails / 30L.

[0013] Further, the breeding density of the juvenile period during the breeding process is 10-30 tails / 30L.

[0014] Further, the final concentration of thyroid hormone T3 in the water body is 0.1 μg / mL.

[0015] Further, the thyroid hormone T3 is diluted with DMSO solution.

[0016] Further, the breeding time of the embryo development period is 72 h.

[0017] Further, the breeding time of the juvenile period is 30 days.

[0018] Further, the breeding light cycle is 14 hours of light: 10 hours of darkness.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application provides a new perspective to examine the role of thyroid hormone in fish color pattern regulation, especially its influence on the development path of non-melanin pigment cells. Based on the above research results, the present application can be applied to:

[0021] Selective breeding of ornamental fish, optimizing body color characteristics by adjusting thyroid hormone levels.

[0022] Development of color management strategies in aquaculture, such as adjusting feed ingredients or water quality conditions to affect thyroid hormone levels, to achieve ideal body color performance.

[0023] Study the interaction between thyroid hormone and other endocrine factors to further understand animal pigments and biological development.

[0024] In summary, the present application firstly confirms the inhibitory effect of thyroid hormone on the development of goldfish red pigment cells and carotenoid accumulation, fills the knowledge gap in this field, and provides important guidance information for future color regulation and genetic improvement of ornamental fish and other aquatic animals. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For the overall diagram of goldfish fry under different hormone treatments during the development in Example 1 (wherein, Figure 1 a-f in the figure are 16h-72h goldfish fry in the control group, Figure 1 g-l in the figure are 16h-72h goldfish fry treated with PTU, Figure 1 m-r in the figure are 16h-72h goldfish fry treated with PTU+T3, Figure 1 s-x in the figure are 16h-72h goldfish fry treated with PTU+TU; the red pigment cells are indicated by white arrows);

[0026] Figure 2 For the enlarged diagram of the trunk of goldfish fry under different hormone treatments in Example 1 (wherein, Figure 2 a-f in the figure are the trunk of goldfish fry treated with PTU for 16h-72h, Figure 2 g-l in the figure are the trunk of goldfish fry treated with PTU+T3 for 16h-72h, Figure 2 m-r in the figure are the trunk of goldfish fry treated with PTU+TU for 16h-72h; the red pigment cells are indicated by white arrows);

[0027] Figure 3 For the diagram of cell number and cell diameter in fry at different development stages in Example 1 (wherein, Figure 3 A is the diagram of red pigment cell number, Figure 3 B is the diagram of red pigment cell diameter);

[0028] Figure 4 For the body color diagram of goldfish treated with thyroid hormone T3 and thyroid hormone inhibitor TU for 30 days in Example 1 (wherein, Figure 4 A kind of con represents the control group, T3 indicates the T3 treatment group, and TU indicates the TU treatment group; a', b', c' are white frame areas in a, b and c, respectively; a'', b'', c'' are head features of the control group, the T3 treatment group and the TU treatment group, respectively; a''', b''', c''' are dorsal fin features of the control group, the T3 treatment group and the TU treatment group, respectively; Figure 4 B is a comparison of T3 content; Figure 4 C is a comparison of T4 content; Figure 4 D is the number of black spots; Figure 4 E is the number of red box melanin cells;Figure 4 F is a comparison of luminance value L; Figure 4 G is a comparison of red-green value a; Figure 4 H is a comparison of yellow-blue value b; Figure 4 I is a comparison of carotenoid content.

[0029] Figure 5 Gene expression related to carotenoid transport and deposition and pteridine synthesis in head skin in Example 1 (wherein the symbols "*" and "**" above the error bar indicate significant differences at the levels of p = 0.05 and p = 0.01, respectively). DETAILED DESCRIPTION

[0030] In order to make the present application better understood, the following examples are given. Obviously, the described examples are only a part of the present application, but not all the examples. Based on the examples in the present application, other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0031] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0032] Example 1

[0033] 1.1 Reagents

[0034] Thyroid hormone T3 (product number: T2877) and thiourea (TU) (product number: T8656) were purchased from Sigma-Aldrich; dimethyl sulfoxide DMSO (product number: A503039) was purchased from Shengong Biotechnology Co., Ltd.; RNA stabilization reagent was purchased from Thermo Scientific; Trizol kit was purchased from Invitrogen.

[0035] 1.2 Drug soaking

[0036] All the embryos, larvae and juvenile fish used in the experiments were hatched and cultured in water tanks (30 liters) on Donghai Island, Zhanjiang City, Guangdong Province. The constant seawater environment temperature was maintained at about 29℃ during the experiment, the light cycle was 14 hours light: 10 hours dark (L:D = 14:10), the water body pH was 7.5-8.0, the salinity was 25‰, and the oxygen content was 6.4±0.2 mg / L. The data in the present application are expressed as mean ± standard error (SE) (n = 3). Two-way ANOVA multiple comparisons were used to analyze the differences in gene expression by using GraphPad Prism 9.0 (GraphPad Software Inc.), and the significance level was P < 0.05.

[0037] In the larval stage of goldfish, the red pigment cells on the skin are gradually covered by melanin. Therefore, in order to observe the development of red pigment cells, 1-phenyl-2-thiourea (PTU) was used to inhibit the formation of melanin.

[0038] After fertilization, all embryos were randomly divided into three groups: PTU control group (normal thyroid function group), PTU + T3 treatment group (hyperthyroidism group) and PTU + TU treatment group (hypothyroidism group). Each group has three replicates, and each replicate has 1000 embryos. In the PTU, PTU + T3 and PTU + TU treatment groups, PTU, T3 and TU are all diluted with an equal amount of DMSO solution, and the embryos in the water tank simultaneously receive PTU with a concentration of 0.003% (200 μM). In the PTU + T3 group, the final concentration of thyroid hormone T3 added to the water body is 0.1 μg / mL. In the PTU + TU group, in order to cause hypothyroidism, TU diluted with DMSO is dissolved in natural seawater, and the final concentration is 0.02%. In the PTU group, an equal amount of DMSO solvent is added. The water body needs to be replaced every day, and PTU, T3 and TU solutions are added every day according to the above operation. The larvae were sampled and photographed at 16, 24, 36, 48, 60 and 72 hpf, respectively. ImageJ software (Rawak Software Inc., Stuttgart, Germany) was used to quantitatively analyze the number and diameter of cells.

[0039] The experimental results show that: Figure 2 (a-f) are 16h-72h goldfish larvae treated with PTU (control group), (g-l) are 16h-72h goldfish larvae treated with PTU + T3, (m-r) are 16h-72h goldfish larvae treated with PTU + TU. From the morphological diagram, it can be found that T3 treatment inhibits the formation of red pigment cells in goldfish at any development period. Figure 3 A is the difference comparison of the number of red pigment cells of 16h-72h goldfish larvae under different treatment conditions. The number of red pigment cells in the T3 group is significantly less than that in the control group and the TU group. Figure 3 B is the comparison of the diameter of red pigment cells of 16h-72h goldfish under different treatment conditions. The diameter of red pigment cells in the T3 group is significantly smaller than that in the other two groups. The experimental results show that thyroid hormone T3 can inhibit the formation of red pigment cells, and at the same time, it can inhibit the diameter of red pigment cells.

[0040] 1.3 Determination of body surface color difference value

[0041] The effects of thyroid hormones and their inhibitors on the red pigment cells of juvenile golden bream were investigated using the same method described above, with an experimental period of 30 days. Juvenile experiment: 60-day-old (dpf) golden bream were selected as model organisms and subjected to a one-month experimental treatment. A control group, a T3 treatment group (final T3 concentration in water was 0.1 μg / mL, T3 to simulate hyperthyroidism), and a TU treatment group (final thiourea concentration in water was 0.02%, TU to simulate hypothyroidism) were set up. As before, T3 and TU were diluted with an equal volume of DMSO solution, and the control group received the same amount of DMSO as the T3 and TU groups. The concentrations of the T3 and TU treatment groups were the same as in the juvenile experiment. Each group had three replicates, with 20 golden bream juveniles in each replicate. The water is changed daily, and the required solution is added daily. The rearing temperature is about 29℃. The fish is fed twice a day with Gongli pomfret fry feed F3, which is purchased from Guangdong Yuequn Marine Biotechnology Co., Ltd. The feed contains ≥48% crude protein and ≥4% crude fat.

[0042] The specific steps are as follows: When the juvenile goldfish were raised from 60 to 90 days old, three experimental fish were randomly selected from each replicate. They were anesthetized with MS-222, their body surface moisture was wiped dry, and the skin color difference value was measured using a colorimeter. Measurement site: the red skin area on the head. Before measurement, the colorimeter was calibrated with a white plate. Measurements were taken twice at each location. During the second measurement, the probe was rotated 180°, and the body color value was recorded. The L* value represents brightness; a larger value indicates a brighter body color. A positive a* value represents a reddish tint, and a negative a* value represents a greenish tint. A positive b* value represents a yellowish tint, and a negative a* value represents a bluish tint. After measurement, red (RS) and black (BS) skin samples were taken from the control group, T3 group, and TU group, stored in an RNA stabilizing reagent for RNA extraction, and stored at -80℃ for further pigment analysis.

[0043] Figure 4 The color change of the goldfish after treatment with T3 in group A was obvious. Figure 4 D showed a significant reduction in the number of black spots on the trunk in the T3 group; however, Figure 4 The results showed that after thyroid hormone signaling was suppressed, the number of melanocytes in the TU group increased significantly. Figure 4 In A, a”, b”, and c” represent the head features of the control group, T3 treatment group, and TU treatment group, respectively. In the T3 group, the red coloration on the head of the goldfish was significantly reduced. Figure 4 In groups a”', b”', and c”' of group A, the red coloration on the back of the goldfish in group T3 was also significantly reduced. Figure 4 G represents the comparison of the red-green color value a of the head skin of the golden coin fish. The redness of the T3 group was significantly lower than that of the control group and the TU treatment group, indicating that thyroid hormone treatment can significantly reduce the redness of the golden coin fish skin.

[0044] 1.4 Determination of thyroid hormones and total carotenoids

[0045] Hormone determination: about 1 g skin of the above cultured to 90 days of goldfish was homogenized, fish T3 and T4 enzyme-linked immunosorbent kit (Shanghai enzyme-linked) was used, according to the kit instructions, T3 and T4 levels of three fish were determined in each group analysis. The effect of thyroid hormone and its inhibitor on the color change of goldfish was explored by the above method, first of all, to prove that these changes are caused by thyroid hormone changes, the thyroid hormones T3 and T4 were measured, and the results were shown in the graph of Figure 4 B, 4C, after T3 treatment, T3 and T4 were significantly increased, while TU group was significantly decreased, indicating that the drug treatment was effective.

[0046] Pigment determination: about 1 g skin of the above cultured to 90 days of goldfish was homogenized, 0.01 g of anhydrous sodium sulfate was added to the red skin tissue of goldfish, and was placed in a 2 mL centrifuge tube, 2 mL of pigment extraction solution (n-hexane: acetone: anhydrous alcohol = 2:1:1, containing 0.01% 2, 6-di-tert-butyl-p-cresol BHT) was added, homogenized for 20 min, then transferred to a 15 mL centrifuge tube, and diluted to 10 mL, and placed in a 4℃ refrigerator for 24 h. 4℃, 6000r / min centrifugation for 10 min, the upper liquid containing pigment was sucked and transferred to a new 15 mL centrifuge tube. The supernatant was washed with saturated NaCl solution for three times until it was neutral, and the total amount of carotenoids was calculated by measuring the absorbance value of the pigment extraction solution at the maximum absorption peak (438 nm) by ultraviolet spectrophotometer. The calculation formula is as follows:

[0047] X = A x K x V / E x M

[0048] In the formula, X is the content of carotenoids (mg / kg), A is the absorbance value, K is the constant 10 4 , V is the volume of extraction solution (mL), E is the molar extinction coefficient (2500), and M is the sample weight (g).

[0049] The effect of thyroid hormone and its inhibitor on the total carotenoids of the red skin of goldfish was explored by the above method, and the results were shown in the graph of Figure 5 I, after inhibiting thyroid hormone, the content of carotenoids increased significantly.

[0050] 1.5 RNA extraction and real-time fluorescent quantitative PCR (qRT-PCR)

[0051] Total RNA of red skin (RS) tissue of control, T3 and TU treated juvenile fish (three tubes for each group) was extracted using Trizol kit according to the manufacturer's instruction. The primers for qRT-PCR were designed from NCBI (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=Blast Home) and listed in Table 1 below.

[0052] Table 1 Primer sequences

[0053]

[0054]

[0055] Using Uni All-in-One First-Strand cDNA Synthesis Super Mix for qRT-PCR for reverse transcription. The expression level of target genes was quantified by qRT-PCR using Green qRT-PCR SuperMix (TransGen, Beijing, China) on Roche LightCycler® 96 System. The thermal cycling program included an initial denaturation at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, 60°C for 15 s, and 72°C for 10 s. The relative expression of target genes was calculated by 2-ΔΔCt method.

[0056] The results are shown in the following figures. After inhibiting thyroid hormone, the expression of carotenoid and pteridine pigment related genes apod, pnpla2, wu:fc46h12, rdh12, stard10, xdh, gch1, abca1, retsat, scarb1, rgs2, bco2, and ttc39b was significantly up-regulated. ​

[0057] ​​The present invention aims to disclose and protect a new scientific discovery that thyroid hormones not only inhibit the formation of melanophores in goldfish (or other potentially affected species), but also inhibit the formation of erythrophores and the accumulation of carotenoids in the body. This discovery is based on the results of a series of rigorous experimental designs, including but not limited to microscopic observation statistics after treatment with thyroid hormones and their inhibitors, skin color difference value determination, spectrophotometric determination of carotenoid concentration, and gene expression studies. These studies show that excessive thyroid hormones can significantly reduce the number of erythrophores and the size of pigment granules, and reduce carotenoid content; while the synthesis of thyroid hormones is inhibited, it will promote the expression of melanophores and related synthesis pathway genes, and increase the level of carotenoids.

[0058] In summary, the present invention provides a completely new perspective to examine the role of thyroid hormones in the regulation of body color patterns in fish, especially its influence on the development path of non-melanin pigment cells. Based on the above research results, the present invention can be applied to:

[0059] Selective breeding of ornamental fish to optimize body color characteristics by adjusting thyroid hormone levels.

[0060] Development of color management strategies in aquaculture, such as adjusting feed composition or water quality conditions to affect thyroid hormone levels to achieve desired body color performance.

[0061] Study the interaction between thyroid hormones and other endocrine factors to further understand animal pigmentation and biological development.

[0062] In summary, the present invention first clearly demonstrates the inhibitory effect of thyroid hormones on the development of erythrophores and the accumulation of carotenoids in goldfish, filling the knowledge gap in this field and providing important guidance for future research on body color regulation and genetic improvement of ornamental fish and other aquatic animals.

[0063] The above-described embodiments are merely preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements to the technical solutions of the present invention made by those skilled in the art shall fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method of regulating the color of an ornamental fish, characterized by, The ornamental fish is Scatophagus argus; the method for regulating the body color of the ornamental fish is to inhibit the development of red pigment cells on the body surface of the ornamental fish and the accumulation of carotenoids, so as to regulate the red body color of the ornamental fish; and the method is as follows: placing the ornamental fish in the embryonic development period after fertilization in the water body, and adding thyroid hormone T3 with a final concentration of 0.05-0.15 μg / mL into the water body, and culturing for 24-96 h; placing the ornamental fish in the juvenile period in the water body, and adding thyroid hormone T3 with a final concentration of 0.05-0.15 μg / mL into the water body, and culturing for 20-40 days.

2. The method of claim 1, wherein, The pH value of the water body for cultivation is 7.5-8.0, and the oxygen content is 6.4±0.2 mg / L.

3. The method of claim 2, wherein, The cultivation temperature is 25-30℃, and the cultivation salinity is 20-30‰.

4. The method of claim 3, wherein, The cultivation density in the embryonic development period is 700-1300 tails / 30 L during the cultivation process.

5. The method of claim 3, wherein, The cultivation density in the juvenile period is 10-30 tails / 30 L during the cultivation process.

6. The method of claim 5, wherein, The final concentration of thyroid hormone T3 in the water body is 0.1 μg / mL.

7. The method of claim 6, wherein, The thyroid hormone T3 is diluted with a DMSO solution.

8. The method of claim 1, wherein, The cultivation time in the embryonic development period is 72 h.

9. The method of claim 1, wherein, The cultivation time in the juvenile period is 30 days.

10. The method of claim 1, wherein, The light cycle for cultivation is 14 hours of light: 10 hours of darkness.

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