Opsin-based industrialized culture illumination method for Oncorhynchus mykiss at different growth stages

Abstract

The invention relates to an opsin-based industrialized culture illumination method for steelhead trout in different growth stages, which is based on physiological characteristics of steelhead trout in different growth stages, adopts an illumination scheme with a specific wavelength, and optimizes growth and health management in a culture process. In the stage that the weight of the steelhead trout is less than 3g, using ultraviolet light with the wavelength of 360-370 nm to irradiate the steelhead trout; when the weight of the hardhead trout ranges from 3 g to 8 g, ultraviolet light and blue light with the wavelength ranging from 430 nm to 445 nm are adopted for jointly irradiating, the ultraviolet light irradiation amount is gradually reduced according to the increase of the weight of the hardhead trout, and the blue light irradiation amount is increased; when the weight of the hardhead trout is larger than 8 g and smaller than 38 g, blue light is used for irradiation; when the weight of the hardhead trout is greater than 38 g, the hardhead trout is irradiated by using red light with the wavelength of 560-580 nm. By accurately controlling the light wavelength and the illumination duration, the growth rate and the immunity of the Oncorhynchus mykiss are improved, and the culture benefits are optimized.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, and in particular to an opsin-based factory farming lighting method for steelhead trout at different growth stages, so as to optimize the factory farming effect thereof and improve the growth rate, immunity and farming efficiency. Background Art

[0002] Salmon and trout are one of the fastest growing and most valuable aquaculture species in the world. [1] , which is rich in a variety of nutrients that are beneficial to human health and is popular among the public [2, 3] With the rapid development of the domestic economy, the demand for salmon and trout is growing, but the domestic aquaculture output is small, and a large number of salmon and trout can only be imported. In recent years, with the continuous improvement of aquaculture technology and aquaculture equipment, the domestic salmon and trout aquaculture output has also increased.

[0003] Steelhead trout is a high-value aquaculture fish with broad market prospects. Its growth and development process are affected by a variety of environmental factors, such as water temperature, light, nutrition, etc. Studies have shown that light colors of different wavelengths have a significant impact on the growth and immunity of fish. At different growth stages of fish, light can affect the physiological functions of fish by regulating the central nervous system and endocrine system.

[0004] Chinese invention patent ZL2021104645551 provides a "light color and light intensity control method for rainbow trout factory farming". This method divides the daily lighting time into five periods, using shorter wavelength blue light, slightly longer wavelength yellow light, longer wavelength red, slightly longer wavelength yellow light, and shorter wavelength blue light. However, the research object of this method, rainbow trout, grows in fresh water for life. However, steelhead trout is an upstream migratory fish, that is, there are two life stages in freshwater and seawater, and there are certain differences in physiological characteristics. The lighting method suitable for rainbow trout is not suitable for steelhead trout. Moreover, its experiment only explored the juvenile stage, without considering the physiological differences in different growth stages, and adopted the same lighting mode, which is not conducive to maximizing the lighting effect. Although this method has noticed that light regulation has various physiological effects on rainbow trout, it only derives the wavelength suitable for rainbow trout through comparative experiments, and lacks the essential research on the mechanism by which light affects rainbow trout. In addition, the method is designed according to the daily rhythm of the light change mode, need to be equipped with three different light sources, and turn on in turn, there is a certain equipment cost, and also has a certain complexity in control and operation.

[0005] At present, domestic salmon and trout farming does not use supplementary lighting based on the physiological characteristics of salmon and trout. Inappropriate supplementary lighting strategies can easily cause problems such as slow growth and development of the fish, susceptibility to disease, bone deformities, and low yields. Existing aquaculture technology usually uses the same light source for irradiation at different growth stages, ignoring the differences in fish's light requirements at different growth stages, and does not select a suitable light environment based on the physiological characteristics of fish, resulting in low growth efficiency and even adverse effects on fish health. Therefore, there is an urgent need for a lighting method for steelhead trout at different growth stages to achieve optimal regulation of its growth and development. Summary of the invention

[0006] The present invention proposes a visual protein-based factory farming lighting method for steelhead trout at different growth stages. According to the characteristics of steelhead trout visual protein in each period, the combined use of ultraviolet light, blue light and red light is precisely controlled to meet the needs of steelhead trout at different growth stages, thereby improving the growth rate, immunity and overall health of steelhead trout.

[0007] A method for industrialized cultivation of steelhead trout at different growth stages based on opsin, characterized in that: 1. For fry weighing less than 3 g, use ultraviolet light with a wavelength of 360 nm-370 nm for irradiation; Purpose: This wavelength of ultraviolet light can enhance the immunity of young fish, promote early development, and reduce the incidence of diseases.

[0008] Implementation: Light intensity was controlled at 30 μmol / m² / s, and the daily exposure time was 12 hours.

[0009] 2. For juveniles weighing 3 g to 8 g, The ultraviolet light with a wavelength of 360 nm-370 nm and the blue light with a wavelength of 430 nm-445 nm are used for irradiation; Purpose: As the weight increases, gradually reduce the amount of ultraviolet light and increase the amount of blue light to promote the development of fish bones and muscles and maintain improved immunity.

[0010] Implementation: The UV light intensity was gradually reduced to 20 μmol / m² / s, and the blue light intensity was gradually increased from 10 μmol / m² / s to 30 μmol / m² / s, and the irradiation time was maintained at 12 hours / day.

[0011] 3. For juveniles weighing 8 g to 38 g, Stop UV irradiation and use blue light with a wavelength of 430 nm-445 nm alone instead; Purpose: Blue light can significantly accelerate the bone and muscle growth of fish at this stage.

[0012] Implementation: The light intensity was controlled at 40 μmol / m² / s and the irradiation time was maintained at 12 hours / day.

[0013] 4. For adult fish weighing more than 38 g, Irradiation was performed using red light with a wavelength of 560 nm-580 nm; Purpose: Red light can promote sexual maturity and gonadal development of steelhead trout at this stage, ensuring their healthy reproductive performance.

[0014] Implementation: The light intensity was 50 μmol / m² / s and the irradiation time was maintained at 12 hours / day.

[0015] Compared with traditional aquaculture technology that mostly uses a single light source, the present invention has the following advantages: Improve growth rate: Based on the changing characteristics of visual proteins in the retina of steelhead trout at different growth stages, light regulation at different growth stages is designed, which is more in line with the growth law of steelhead trout and significantly improves the growth rate of steelhead trout.

[0016] Enhance immunity: Light of different wavelengths can effectively enhance the immunity of steelhead trout and reduce the incidence of diseases.

[0017] Economic benefits: This lighting method has broad application prospects in factory farming and can significantly improve farming efficiency.

[0018] Cost advantage: Single light color is easier to control and more convenient to implement.

[0019] In summary, the present invention provides a lighting method for factory farming of steelhead trout at different growth stages. According to the physiological characteristics of the changes in visual proteins in the retina of steelhead trout at different growth stages, ultraviolet light, blue light and red light of different wavelengths are used at different growth stages to effectively improve the growth rate, immunity and health level of steelhead trout. This method can not only significantly improve the farming efficiency, but also has a wide range of application prospects, especially in the large-scale industrial farming of steelhead trout, with significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of light regulation for steelhead trout in different growth stages DETAILED DESCRIPTION

[0021] The basic method of the present invention is to improve the growth and immune performance of steelhead trout by controlling the light exposure mode. The light exposure and body weight change curve is as follows: Figure 1 shown.

[0022] 1: Lighting method for the stage less than 3 g When the weight of steelhead trout is less than 3 g, ultraviolet light with a wavelength of 360 nm-370 nm is used for irradiation, and the light intensity is controlled at 30 μmol / m² / s, and the irradiation time is 12 hours per day. This method can significantly enhance the immunity of steelhead trout and reduce the occurrence of diseases.

[0023] 2: Lighting method for 3 g-8 g stage When the steelhead trout weighed 3 g to 8 g, they were irradiated with both ultraviolet light and blue light. The intensity of ultraviolet light was gradually reduced to 20 μmol / m² / s, while the intensity of blue light was gradually increased from the initial 10 μmol / m² / s to 30 μmol / m² / s. The irradiation time was maintained at 12 hours per day. Through this adjustment, the immunity and growth rate of steelhead trout were improved simultaneously.

[0024] 3: Lighting method for 8 g-38 g stage When the weight of steelhead trout was greater than 8 g but less than 38 g, ultraviolet light irradiation was stopped and replaced with blue light with a wavelength of 430nm-445nm, with the light intensity controlled at 40 μmol / m² / s and the irradiation time for 12 hours per day. Blue light irradiation significantly promoted the bone development and growth rate of steelhead trout at this stage.

[0025] 4: Lighting method for stages above 38 g When the weight of steelhead trout is greater than 38 g, red light with a wavelength of 560 nm-580 nm is used for irradiation, with a light intensity of 50 μmol / m² / s and an irradiation time of 12 hours / day. Red light at this stage can promote the sexual maturity and gonadal development of steelhead trout, ensure its reproductive health and overall growth quality, and provide guarantee for the continuation of breeding.

[0026] Implementation process The invention adopts illumination schemes of different wavelengths according to the physiological characteristics of changes in visual proteins in the retina of steelhead trout at different growth stages.

[0027] Studies on fish opsins have been [4] We found that the opsins in the retina of steelhead trout include RH1 in rods and SWS1, SWS2, RH2 and M / LWS in cones, which can sense light intensity, ultraviolet light, blue light, green light and red light respectively. As receptors for fish to respond to light environment, opsins affect their physiological functions by regulating the central nervous system and endocrine system. A suitable light environment is of great significance to improving fish welfare.

[0028] There has also been some research on the development of fish retina [5]However, the existing research is not comprehensive and systematic, especially the visual protein expression and visual pigment absorption spectral characteristics of steelhead trout at different growth stages need to be further improved.

[0029] Currently, most studies on steelhead trout opsins focus on small-sized fish, and no systematic studies have been conducted throughout the developmental stages. [6, 7] .

[0030] The expression of rod cells in fish in different water layers is significantly different, and the expression of rod cells in deep-sea fish is significantly increased [8] At different developmental stages of fish, due to changes in the light environment in which they live, the expression of opsin cones also varies significantly. [5, 9, 10] .

[0031] Visual pigments are the core of the fish visual system, which determines the maximum absorbance wavelength (λmax) that fish are most sensitive to. There are large differences in the spectral adaptability of fish at different developmental stages. It is more accurate and reliable to determine the optimal spectral range and peak value of steelhead trout based on the visual pigment absorption spectrum, which is more suitable for its growth needs.

[0032] The studies on the effects of different spectra (light colors) on the physiology and biochemistry of rainbow trout reported so far have only limited the spectral wavelength range and have not set the spectral peak value based on the absorption spectrum characteristics of fish visual pigments. This may be the reason for the inconsistent research results. [11-16] .

[0033] Therefore, the present invention aims to address the above issues and explore the morphological structure and distribution characteristics of opsin in the retina of steelhead trout at different growth stages from the following two aspects.

[0034] Research content

[0035] (1) Expression characteristics of opsin genes in steelhead trout at different life history stages Real-time fluorescence quantitative PCR (RT-qPCR) was used to determine the expression characteristics of five opsins, including RH1 in the retinal rods, SWS1, SWS2, RH2 and M / LWS in the retinal cones of steelhead trout at the larvae, juveniles, young fish and adults, to explore the differences in the requirements of steelhead trout for light intensity and light color at different life history stages. (2) Absorption spectra of visual pigments of steelhead trout at different life history stages

[0036] Microspectrophotometry (MSP) was used to measure the maximum absorbance wavelengths (λ) of the visual pigments of five opsins in the retina of steelhead trout at the larval, juvenile, young and adult stages. max ), and used the visual photopigment modeling method based on atomic molecular dynamics simulation (MD) for verification analysis to explore the absorption spectral range and peak value of the optimal light color in different life history stages of steelhead trout.

[0037] Research Methods

[0038] (1) Expression characteristics of opsin genes in steelhead trout Real-time fluorescence quantitative PCR (RT-qPCR) was used to determine the expression of opsin genes in the retina of steelhead trout at the mRNA level. Primer Premier 6.0 software was used to design the expression primers of the internal reference genes RH1, SWS1, SWS2, RH2, and M / LWS of steelhead trout, and 18S rRNA, β-actin, ubiquitin, EF-1α, and β-tubulin were used as internal reference genes for amplification. The mRNA expression characteristics of retinal RH1, SWS1, SWS2, RH2, and M / LWS opsin synthesis in steelhead trout at different life history stages (larvae, juveniles, young fish, and adults) were compared.

[0039] (2) Absorption spectrum of steelhead trout visual pigments

[0040] The absorption spectra of steelhead trout visual pigments were determined by microspectrophotometry (MSP) and atomistic molecular dynamics simulation-based visual pigment modeling (MD).

[0041] The MSP method for determining visual pigment absorption spectra refers to Kondrashev (2022). After the steelhead trout were adapted to a dark environment for 2-3 h, they were anesthetized with 300 mg / L ethyl m-aminobenzoate methanesulfonate (MS-222, Sigma Chemicals Inc., USA) to cause death. The eyeball tissue was then removed in a dim red light environment, and the retina was extracted and placed in a phosphate-buffered saline solution (PBS; Sigma-Aldrich) containing 6.0% sucrose. Part of the retina was cut and placed on a glass slide. After an appropriate amount of PBS buffer was dropped, a coverslip was covered and sealed with high vacuum silicone grease. The maximum absorbance wavelength (λ max ).

[0042] in conclusion

[0043] When the body weight of steelhead trout was 3 g, the content of SWS1 protein in the retina was the highest, and the λmax was between 360 nm and 370 nm; when the body weight of steelhead trout was 3 g-8 g, the content of SWS1 protein in the retina gradually decreased, while SWS2 protein showed a gradual increasing trend, the λmax of SWS2 protein was between 430 nm and 445 nm, and the RH1 protein showed a decreasing trend; when the body weight of steelhead trout was 8 g-38 g, the SWS1 protein disappeared in the retina, while the SWS2 protein gradually rose to the highest point, and the RH1 protein gradually increased, and was higher than the expression level of steelhead trout when the body weight was 3 g; when the body weight of steelhead trout was greater than 38 g, the SWS2 protein decreased, while the M / LWS protein continued to be high, the λmax of the M / LWS protein was between 560 nm and 580 nm, and the RH1 protein continued to increase and then stabilized, and was higher than the expression level of steelhead trout when the body weight was 8 g-38 g.

[0044] By exploring the expression of visual proteins and the absorption spectrum characteristics of visual pigments in the retina of steelhead trout at different developmental stages, the present invention established a lighting supplement strategy for steelhead trout at different growth stages.

[0045] References [1] Landazuri-Tveteraas, U., et al., 2021. Salmon trout, theforgotten cousin? Aquaculture Economics & Management, 25(2), 159-176. [2] Golden, CD, et al., 2021. Aquatic foods to nourish nations,Nature, 598, 315-320. [3] Hart, B., Schurr, R., et al., 2021. Digestibility of Schizochytrium sp. whole cell biomass by Atlantic salmon ( Salmo salar ),Aquaculture, 533, 736156. [4] Liu Chuwu et al., 2015. Research progress on fish visual proteins, Oceanology and Limnology, 46, 1564-1570. [5] Allison, W.T., et al., 2006. Degeneration and regeneration ofultraviolet cone photoreceptors during development in rainbow trout, Journalof Comparative Neurology, 499(5), 702-715. [6] Allison, W.T., et al., 2003. Ontogeny of ultraviolet-sensitivecones in the retina of rainbow trout ( Oncorhynchus mykiss ), Journal ofComparative Neurology, 461(3), 294-306. [7] Cheng, C.L., et al., 2006. Photoreceptor layer of salmonidfishes: transformation and loss of single cones in juvenile fish, Journal ofComparative Neurology, 495(2), 213-235. [8] Musilova, Z., et al., 2019. Vision using multiple distinct rodopsins in deep-sea fishes, Science, 364(6440), 588-592. [9] Deutschlander, et al., 2001. Functional mapping of ultravioletphotosensitivity during metamorphic transitions in a salmonid fish, Oncorhynchus mykiss , Journal of Experimental Biology, 204(14), 2401-2413.

[10] Cheng, C.L., Novales Flamarique, I., 2004. Opsin expression: newmechanism for modulating colour vision, Nature, 428(6980), 279-280.

[11] Guller, U., et al., 2020. Effects of different LED light spectraon rainbow trout ( Oncorhynchus mykiss ): in vivo evaluation of the antioxidantstatus, Fish Physiology and Biochemistry, 46(6), 2169-2180.

[12] Heydarnejad, M.S., et al., 2013. Influence of light colours ongrowth and stress response of rainbow trout ( Oncorhynchus mykiss ) underlaboratory conditions, Journal of Animal Physiology and Animal Nutrition, 97(1), 67-71.

[13] Karakatsouli, N., et al., 2008. Effects of light spectrum ongrowth and stress response of rainbow trout Oncorhynchus mykiss reared underrecirculating system conditions, Aquacultural Engineering, 38(1), 36-42.

[14] Karakatsouli, N., et al., 2007. Effects of light spectrum ongrowth and physiological status of gilthead seabream Sparus aurata andrainbow trout Oncorhynchus mykissreared under recirculating systemconditions, Aquacultural Engineering, 36(3), 302-309.

[15] Luchiari, A.C., Pirhonen, J., 2008. Effects of ambient colour oncolour preference and growth of juvenile rainbow trout Oncorhynchus mykiss (Walbaum), Journal of Fish Biology, 72(6), 1504-1514.

[16] Ma, S.S.,et al., 2023. Influence of daily rhythmic light spectraand intensity changes on the growth and physiological status of juvenilesteelhead trout ( Oncorhynchus mykiss ), Frontiers in Marine Science 10,1116719.

Claims

1. A method for industrialized cultivation of steelhead trout at different growth stages based on opsin, characterized in that:

1. For fry weighing less than 3 g, use ultraviolet light with a wavelength of 360 nm-370 nm for irradiation; 2. For juvenile fish weighing 3 g to 8 g, use ultraviolet light with a wavelength of 360 nm-370 nm and blue light with a wavelength of 430 nm-445 nm; 3. For juvenile fish weighing between 8 g and 38 g, stop UV irradiation and use blue light with a wavelength of 430 nm-445 nm alone; 4. For adult fish weighing more than 38 g, use red light with a wavelength of 560 nm-580 nm for irradiation.

2. The method for industrialized cultivation of steelhead trout at different growth stages based on opsin according to claim 1, characterized in that: During the larval stage, the light intensity was controlled at 30 μmol / m² / s and the daily exposure time was 12 hours.

3. The method for industrialized cultivation of steelhead trout at different growth stages based on opsin according to claim 2, characterized in that: During the juvenile stage, the UV light intensity was reduced to 20 μmol / m² / s, and the blue light intensity was gradually increased from 10 μmol / m² / s to 30 μmol / m² / s, and the exposure time was maintained at 12 hours / day.

4. The method for industrialized cultivation of steelhead trout at different growth stages based on opsin according to claim 3, characterized in that: During the larval stage, the light intensity was controlled at 40 μmol / m² / s and the daily exposure time was 12 hours.

5. The method for industrialized cultivation of steelhead trout at different growth stages based on opsin according to claim 4, characterized in that: During the adult stage, the light intensity was 50 μmol / m² / s and the exposure time was maintained at 12 hours / day.

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