Method for regulating body color of ornamental fish

Abstract

The invention discloses a method for regulating the body color of ornamental fish, and belongs to the technical field of aquaculture. According to the method disclosed by the invention, the aquarium fishes in the fry stage and the juvenile stage are soaked with thyroid hormone, so that the excessive thyroid hormone can obviously reduce the number of haematochrome cells and the size of pigment particles of the aquarium fishes in different stages, and reduce the content of carotenoid; when the synthesis of thyroid hormone is inhibited, gene expression of melanocytes and related synthetic pathways can be promoted, and meanwhile, the carotenoid level is increased. The inhibition effect of thyroid hormone on scatophagus argus haematochrome cell development and carotenoid accumulation is determined for the first time, the knowledge blank in the field is filled, and important guidance information is provided for body color regulation and genetic improvement of aquarium fish and other aquatic animals in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture, and in particular relates to a method for regulating the body color of ornamental fish. Background Art

[0002] Thyroid hormones (TH), including 3,5,3′-L-triiodothyronine (T3), have long been considered key factors in regulating the growth, development, and metabolism of organisms. In fish, thyroid hormones are essential for the metamorphosis process and are believed to inhibit the differentiation and distribution of melanocytes and promote the differentiation of xanthophyll cells and iridophyll cells. However, as red is an important economic trait in aquatic animals, there are relatively few studies on how thyroid hormones affect red pigment cells, especially the lack of understanding of their specific mechanisms for carotenoid metabolism. This knowledge gap limits our more comprehensive understanding of fish body color changes 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 mechanism 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 the order Perciformes, family Scatophagus, genus Scatophagus, commonly known as golden drum. As a famous, special, and valuable marine economic fish in the south, Scatophagus is one of the valuable aquarium fish. At the same time, Scatophagus is delicious, so it is widely favored by consumers. Scatophagus has erythrocytes in the embryonic stage and forms bright red patches on the dorsal side in the late embryonic stage. It is an ideal model fish for studying the regulation of thyroid hormones on erythrocytes and carotenoids. Summary of the invention

[0004] The present invention discloses a new function of thyroid hormone (TH), especially 3,5,3′-L-triiodothyronine (T3), in regulating the body color pattern of ornamental fish, namely, its inhibitory effect on the development of erythrocytes and the accumulation of carotenoids. In addition, the present invention also relates to an application method for controlling or changing the body color of ornamental fish by using this discovery.

[0005] The present invention provides the application of thyroid hormone in a product for improving fish body color. The present invention clarifies for the first time the inhibitory effect of thyroid hormone on the development of erythrocytes and the accumulation of carotenoids in golden croaker, filling the knowledge gap in this field and providing important guiding information for the future body color regulation and genetic improvement of ornamental fish and other aquatic animals.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for regulating the body color of ornamental fish, the method comprising:

[0008] Place the ornamental fish in the embryonic development stage after fertilization in a water body, add thyroid hormone T3 with a final concentration of 0.05-0.15 μg / mL to the water body, and culture for 24-96 hours;

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

[0010] Furthermore, the pH value of the aquaculture water is and the oxygen content is 6.4±0.2 mg / L.

[0011] Furthermore, the aquaculture temperature is 25-30°C and the aquaculture salinity is 20-30‰.

[0012] Furthermore, in the breeding process, the breeding density during the embryonic development period is 700-1300 tails / 30L.

[0013] Furthermore, in the breeding process, the breeding density of juvenile fish is 10-30 fish / 30L.

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

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

[0016] Furthermore, the embryonic development period culture time is 72 hours.

[0017] Furthermore, the juvenile fish culture period is 30 days.

[0018] Furthermore, the culture light cycle is 14 hours light: 10 hours dark.

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

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

[0021] Selective breeding of ornamental fish to optimize body color characteristics by regulating thyroid hormone levels.

[0022] Development of color management strategies in aquaculture, such as adjusting feed composition or water conditions to influence thyroid hormone levels to achieve desired color expression.

[0023] Study the interaction between thyroid hormones and other endocrine factors to gain further insight into animal pigmentation and biological development.

[0024] In summary, the present invention has clarified for the first time the inhibitory effect of thyroid hormone on the development of erythrocytes and the accumulation of carotenoids in golden croaker, filling the knowledge gap in this field and providing important guiding information for the future body color regulation and genetic improvement of ornamental fish and other aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an overall diagram of the development of goldfish larvae under different hormone treatments in Example 1 (wherein, Figure 1 AF is the control group of 16h-72h old golden croaker fry, Figure 1 The gl in the middle is the fry of golden croaker treated with PTU for 16h-72h. Figure 1 The middle mr is the golden croaker fry treated with PTU+T3 for 16h-72h. Figure 1 sx in the middle are golden croaker larvae treated with PTU+TU for 16h-72h; red pigment cells are indicated by white arrows);

[0026] Figure 2 This is a partial enlarged view of the trunk of the golden croaker larvae under different hormone treatments in Example 1 (wherein, Figure 2 Figures a and b are photos of the trunk of golden croaker fry treated with PTU for 16h-72h. Figure 2 The middle gl is a picture of the trunk of a golden croaker fry treated with PTU+T3 for 16h-72h. Figure 2 The middle mr is a picture of the trunk of golden croaker larvae treated with PTU+TU for 16h-72h; the red pigment cells are indicated by white arrows);

[0027] Figure 3 Figure 1 shows the number and diameter of cells in larvae at different developmental stages (wherein, Figure 3 A is the number of red pigment cells. Figure 3 B is the diameter of red pigment cells);

[0028] Figure 4 This is the body color diagram of the golden croaker after being treated with thyroid hormone T3 and thyroid hormone inhibitor TU for 30 days in Example 1 (wherein, Figure 4 Acon represents the control group, T3 represents the T3 treatment group, and TU represents the TU treatment group; a', b', c' are the white frame areas in a, b, and c respectively; a", b", c" are the head features of the control group, T3 treatment group, and TU treatment group respectively; a"', b"', c"' are the dorsal fin features of the control group, T3 treatment group, and TU treatment group respectively; Figure 4 B is the comparison of T3 content; Figure 4 C is the comparison of T4 content; Figure 4 D is the number of black spots; Figure 4 E is the number statistics of melanocytes in the red frame; Figure 4 F is the comparison of the brightness value L; Figure 4 G is the comparison of redness and greenness value a; Figure 4 Comparison of H yellow-blue value b; Figure 4 Comparison of carotenoid content in vitro).

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

[0030] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] Example 1

[0033] 1.1 Reagents

[0034] Thyroid hormone T3 (Product No.: T2877) and thiourea (TU) (Product No.: T8656) were purchased from Sigma-Aldrich; dimethyl sulfoxide DMSO (Product No.: A503039) was purchased from Sangon Biotechnology Co., Ltd.; RNA stabilization reagent was purchased from Thermo Scientific; and Trizol kit was purchased from Invitrogen.

[0035] 1.2 Drug immersion

[0036] All the experimental goldfish embryos, fry and juveniles were hatched and cultured in a water tank (30 liters) on Donghai Island, Zhanjiang City, Guangdong Province. During the experiment, the constant seawater environment temperature was maintained at about 29°C, the photoperiod was 14 hours of light: 10 hours of darkness (L:D=14:10), the water pH was 7.5-8.0, the salinity was 25‰, and the oxygen content was 6.4±0.2mg / L. In the present invention, the data are represented by mean±standard error (SE) (n=3). Two-way ANOVA multiple comparisons were performed using GraphPad Prism 9.0 (GraphPad Software Inc.) to analyze the differences in gene expression, and the significance level was P<0.05.

[0037] Larvae experiment: In the larvae stage of the golden coin, the erythrocytes on the skin are gradually covered with melanin. Therefore, in order to observe the development of erythrocytes, 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 had three replicates, and each replicate had 1000 embryos. In the PTU, PTU+T3 and PTU+TU treatment groups, PTU, T3 and TU were diluted with equal amounts of DMSO solution, and the embryos in the water tank received PTU at a concentration of 0.003% (200μM) at the same time. In the PTU+T3 group, the final concentration of thyroid hormone T3 after adding to the water was 0.1μg / mL. In the PTU+TU group, in order to induce hypothyroidism, TU diluted with DMSO was dissolved in natural seawater to a final concentration of 0.02%. In the PTU group, an equal amount of DMSO solvent was added. The water was changed every day, and PTU, T3 and TU solutions were added every day in the same way as above. The embryos were cultured to form larvae at 16 hpf, and the larvae were sampled and photographed at 16, 24, 36, 48, 60, and 72 hpf. The cell number and diameter were quantitatively analyzed using ImageJ software (Rawak Software Inc., Stuttgart, Germany).

[0039] The experimental results show that: Figure 2 (af) are PTU-treated (control group) 16h-72h larvae, (gl) are PTU+T3-treated larvae 16h-72h larvae, and (mr) are PTU+TU-treated larvae 16h-72h larvae. From the morphological images, it can be found that T3 treatment inhibits the formation of red pigment cells in larvae at any developmental stage. Figure 3 A is a comparison of the number of erythrocytes in golden croaker larvae at the 16h-72h stage under different treatment conditions. The number of erythrocytes in the T3 group was significantly less than that in the control group and the TU group. Figure 3 B is a comparison of the diameter of erythrocytes of the golden croaker under different treatment conditions at the 16h-72h stage. The diameter of erythrocytes in the T3 group was significantly smaller than that in the other two groups. The experimental results show that thyroid hormone T3 inhibits the formation of erythrocytes and at the same time, inhibits the diameter of erythrocytes.

[0040] 1.3 Determination of body surface color difference

[0041] The same method as above was used to explore the effects of thyroid hormones and their inhibitors on the erythrocytes of juvenile golden croakers, and the experimental time was 30 days. Juvenile fish experiment: Golden croakers aged 60 days (dpf) were selected as model organisms for a one-month experimental treatment. A control group, a T3 treatment group (the final concentration of T3 in the water was 0.1 μg / mL, and T3 was used to simulate the state of hyperthyroidism) and a TU treatment group (the final concentration of thiourea in the water was 0.02%, and TU was used to simulate the state of hypothyroidism) were set up. As in the previous article, T3 and TU were diluted with equal amounts of DMSO solution, and the control group was added with the same amount of DMSO as the T3 group and TU group. The concentrations of the T3 treatment group and the TU treatment group were the same as those of the fry experiment. Each group had three replicates, and 20 golden croaker juveniles were placed in each replicate. The water was replaced every day, and the required solution was added every day. The breeding temperature was about 29°C. The fish were fed twice a day. The feed was Gongli Pomfret Fry Feed F3 purchased from Guangdong Yuequn Marine Biotechnology Co., Ltd. The crude protein content of the feed was ≥48%, and the crude fat content was ≥4%.

[0042] The specific steps are as follows: When the juvenile goldfish were raised from 60 days to 90 days, 3 experimental fish were randomly selected for each replicate, anesthetized with MS-222, the body surface moisture was wiped off, and the skin color difference value was measured with a colorimeter. Measurement site: red skin area at the head. The colorimeter was calibrated with a white plate before measurement, and each position was measured twice. The second measurement probe was rotated 180° to record the body color value. The L* value represents brightness, and the larger the value, the brighter the body color; a* value with a positive number represents reddishness, and a negative number represents greenishness; b* value with a positive number represents yellowishness, and a negative number represents bluishness. After the measurement, red skin (RS) and black skin (BS) samples of the control group, T3 group, and TU group were taken, stored in RNA stabilization reagent to extract RNA, and stored at -80°C for further pigment analysis.

[0043] Figure 4 In A, the body color of the golden coin fish changed significantly after being treated with T3. Figure 4 D shows that the number of dark spots on the trunk of the T3 group was significantly reduced, however, Figure 4 E The results showed that after the thyroid hormone signal was inhibited, the number of melanocytes in the TU group increased significantly. Figure 4 a”, b”, and c” in A are the head features of the control group, T3 treatment group, and TU treatment group, respectively. The red coloration of the head of the golden fish in the T3 group was significantly weakened. Figure 4 In groups a”', b”', and c”' in A, the red coloration on the back of the golden croaker in group T3 was also significantly weakened. Figure 4 G is a comparison of the redness and greenness values ​​a of the head skin of golden croaker. 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 croaker skin.

[0044] 1.4 Determination of thyroid hormone and total carotenoids

[0045] Hormone determination: Take about 1g of skin from the above-mentioned goldfish cultured for 90 days and homogenize it. Use fish T3 and T4 ELISA kits (Shanghai ELISA) according to the instructions of the kit to determine the T3 and T4 levels of three fish in each group. The above method was used to explore the effects of thyroid hormones and their inhibitors on the body color changes of goldfish. First, to confirm that these changes are caused by changes in thyroid hormones, thyroid hormones T3 and T4 were measured. The results are shown in the figure below. Figure 4 As shown in B and 4C, after T3 treatment, both T3 and T4 increased significantly, while those in the TU group decreased significantly, indicating that the drug treatment was effective.

[0046] Pigment determination: Take about 1g of the skin of the above-mentioned goldfish cultured for 90 days and homogenize it. Add 0.01g of anhydrous sodium sulfate to the red skin tissue of the goldfish, put it in a 2mL centrifuge tube, add 2mL of pigment extract (n-hexane: acetone: anhydrous alcohol = 2:1:1, containing 0.01% 2,6-di-tert-butyl-p-cresol BHT) and homogenize for 20 minutes, then transfer it to a 15mL centrifuge tube, adjust the volume to 10mL, and put it in a 4℃ refrigerator for 24 hours. Centrifuge at 4℃, 6000r / min for 10 minutes, absorb the upper layer containing the pigment, and transfer it to a new 15mL centrifuge tube. Wash the supernatant three times with a saturated NaCl solution until it is neutral. Measure the absorbance of the pigment extract at the maximum absorption peak (438nm) with an ultraviolet spectrophotometer to calculate the total amount of carotenoids. The calculation formula is as follows:

[0047] X=A×K×V / E×M

[0048] Where X is the carotenoid content (mg / kg), A is the absorbance value, and K is a constant 10 4 , V is the volume of the extract (mL), E is the molar extinction coefficient (2500), and M is the sample weight (g).

[0049] The above method was used to explore the effects of thyroid hormone and its inhibitor on the total carotenoids in the red skin of golden croaker. The results are shown in the figure below. Figure 4 As shown in Figure I, carotenoid content increased significantly after thyroid hormone inhibition.

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

[0051] According to the manufacturer's instructions, the total RNA of the red skin (RS) tissue of the control group, T3 and TU treatment groups obtained above was extracted using the Trizol kit (three tubes per group). The primers designed for qRT-PCR were from NCBI (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=Blast Home), and the designed primers are shown in Table 1 below.

[0052] Table 1 Primer sequences

[0053]

[0054]

[0055] use Reverse transcription was performed using Uni All-in-One First-Strand cDNA Synthesis Super Mixfor qRT-PCR. Green qRT-PCR SuperMix (TransGen, Beijing, China) was purchased from Roche The expression level of the target gene was quantified by qRT-PCR on the 96 system. The thermal cycle program included initial denaturation at 94°C for 30 seconds, followed by 40 cycles of 94°C for 5 seconds, 60°C for 15 seconds, and 72°C for 10 seconds. The relative expression of the target gene was calculated using the 2-ΔΔCt method.

[0056] The above method was used to explore the effects of thyroid hormone and its inhibitor on the expression of genes related to the red skin, carotenoids and pteridine pigments of golden fish. The results are shown in the figure below. Figure 5 After inhibiting thyroid hormone, the expression levels of carotenoid and pteridine pigment-related genes apod, pnpla2, wu:fc46h12, rdh12, stard10, xdh, gch1, abca1, retsat, scarb1, rgs2, bco2, and ttc39b were significantly upregulated.

[0057] The present invention aims to disclose and protect a new scientific discovery, that is, thyroid hormone not only inhibits the production of melanocytes in golden croaker (or other potentially affected species), but also inhibits the formation of erythrocytes and the accumulation of carotenoids in the body. This discovery is based on the results obtained from a series of rigorous experimental designs, including but not limited to microscopic observation statistics after treatment with thyroid hormone and its inhibitors, determination of skin color difference values, determination of carotenoid concentration by spectrophotometry, and gene expression studies. These studies have shown that excessive thyroid hormone can significantly reduce the number of erythrocytes and the size of pigment granules, and reduce the content of carotenoids; when thyroid hormone synthesis is inhibited, it will promote the expression of melanocytes and related synthesis pathway genes, while increasing carotenoid levels.

[0058] In summary, the present invention provides a new perspective to examine the role of thyroid hormone in the regulation of fish body color pattern, especially its impact 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 regulating thyroid hormone levels.

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

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

[0062] In summary, the present invention has clarified for the first time the inhibitory effect of thyroid hormone on the development of erythrocytes and the accumulation of carotenoids in golden croaker, filling the knowledge gap in this field and providing important guiding information for the future body color regulation and genetic improvement of ornamental fish and other aquatic animals.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for regulating the body color of ornamental fish, characterized in that: The method is: Place the ornamental fish in the embryonic development stage after fertilization in a water body, add thyroid hormone T3 with a final concentration of 0.05-0.15 μg / mL to the water body, and culture for 24-96 hours; Juvenile ornamental fish are placed in water, to which thyroid hormone T3 having a final concentration of 0.05-0.15 μg / mL is added, and cultured for 20-40 days.

2. The method according to claim 1, characterized in that The pH value of the aquaculture water is 7.5-8.0, and the oxygen content is 6.4±0.2 mg / L.

3. The method according to claim 2, characterized in that The culture temperature is 25-30°C and the culture salinity is 20-30‰.

4. The method according to claim 3, characterized in that The breeding process has a breeding density of 700-1300 fish / 30L during the embryonic development period.

5. The method according to claim 3, characterized in that: During the breeding process, the breeding density of juvenile fish is 10-30 fish / 30L.

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

7. The method according to claim 6, characterized in that The thyroid hormone T3 is diluted with DMSO solution.

8. The method according to claim 1, characterized in that: The embryonic development period culture time is 72 hours.

9. The method according to claim 1, characterized in that: The juvenile fish culture time is 30 days.

10. The method according to claim 1, characterized in that The culture light cycle is 14 hours light: 10 hours dark.

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