Illumination method for on-growing of different growth stages of oncorhynchus mykiss based on opsin

Abstract

This invention employs a photoluminescence method for intensive steelhead trout farming at different growth stages based on opsin. This method utilizes specific wavelengths of light to optimize growth and health management during the farming process, taking into account the physiological characteristics of steelhead trout at different growth stages. When steelhead trout weigh less than 3 g, ultraviolet light with a wavelength of 360 nm–370 nm is used. When the weight is between 3 g and 8 g, ultraviolet light and blue light with a wavelength of 430 nm–445 nm are used together, with the ultraviolet light intensity gradually decreasing and the blue light intensity increasing as the weight increases. When the weight is greater than 8 g but less than 38 g, only blue light is used. When the weight is greater than 38 g, red light with a wavelength of 560 nm–580 nm is used. This invention improves the growth rate and immunity of steelhead trout and optimizes farming efficiency by precisely controlling the light wavelength and duration.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, specifically to a method for intensive farming of hardhead trout based on opsin, targeting different growth stages, to optimize intensive farming results and improve growth rate, immunity, and farming efficiency. Background Technology

[0002] Salmon and trout are among the fastest-growing and most valuable aquaculture species in the world. [1] It is rich in a variety of nutrients that are beneficial to human health and is highly favored by the public. [2, 3] With the rapid development of China's economy, the demand for salmon and trout has been increasing, but domestic aquaculture production is small, and a large amount of salmon and trout can only be imported. In recent years, with the continuous improvement of aquaculture technology and equipment, domestic salmon and trout aquaculture production has also been increasing.

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

[0004] Chinese invention patent ZL2021104645551 discloses a method for controlling light color and intensity in rainbow trout factory farming. This method divides the daily light exposure into five periods, using shorter wavelength blue light, slightly longer wavelength yellow light, longer wavelength red light, slightly longer wavelength yellow light, and shorter wavelength blue light respectively. However, the rainbow trout studied in this method live their entire lives in freshwater. Hardhead trout, on the other hand, are anadromous fish, meaning they have both freshwater and saltwater life stages, resulting in physiological differences. The lighting method suitable for rainbow trout is not suitable for hardhead trout. Furthermore, the experiment only investigated the juvenile stage, failing to consider the physiological differences at different growth stages, and using the same lighting pattern is not conducive to maximizing the lighting effect. While this method acknowledges the various physiological effects of light regulation on rainbow trout, it merely determines the suitable wavelength for rainbow trout through comparative experiments, lacking a fundamental study of the mechanisms by which light affects rainbow trout. In addition, the light-changing method designed according to the daily rhythm requires three different light sources that are turned on in turn, which has certain equipment costs and is also somewhat complicated in terms of control and operation.

[0005] Currently, salmon and trout farming in China does not utilize supplemental lighting based on the physiological characteristics of salmon and trout. Inappropriate lighting strategies can easily lead to problems such as slow growth and development, increased susceptibility to disease, skeletal deformities, and low yields. Existing aquaculture techniques typically use the same light source for different growth stages, ignoring the varying light requirements of fish at different stages and failing to select suitable light environments based on their physiological characteristics. This results in low growth efficiency and even adverse effects on fish health. Therefore, there is an urgent need for a lighting method tailored to the different growth stages of hardhead trout to achieve optimal regulation of their growth and development. Summary of the Invention

[0006] This invention proposes a lighting method for factory farming of hardhead trout at different growth stages based on opsin. According to the characteristics of hardhead trout opsin at different stages, the method precisely controls the combination of ultraviolet, blue and red light to meet the needs of hardhead trout at different growth stages, thereby improving the growth rate, immunity and overall health of hardhead trout.

[0007] A method for intensive farming of hardhead trout at different growth stages based on opsin, characterized by: 1. For larvae weighing less than 3 g, irradiate with ultraviolet light with a wavelength of 360 nm-370 nm; Objective: Ultraviolet light of this wavelength can enhance the immunity of juvenile fish, promote early development, and reduce the incidence of diseases.

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

[0009] 2. For juvenile fish weighing between 3 g and 8 g, Irradiation was performed using ultraviolet light with a wavelength of 360 nm-370 nm and blue light with a wavelength of 430 nm-445 nm. Objective: To gradually reduce the amount of ultraviolet light and increase the amount of blue light as the fish gain weight, in order to promote the development of the fish's bones and muscles and maintain enhanced immunity.

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

[0011] 3. For juvenile fish weighing between 8 g and 38 g, Stop ultraviolet light irradiation and switch to irradiation with blue light with a wavelength of 430 nm-445 nm; Objective: Blue light can significantly accelerate the growth of bones and muscles in 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 over 38 g, Irradiate with red light with a wavelength of 560 nm-580 nm; Objective: Red light at this stage can promote sexual maturation and gonadal development in hardhead trout, 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 techniques that mostly use a single light source, this invention has the following advantages: Improving growth rate: Based on the changes in opsin in the retina of hardhead trout at different growth stages, light regulation for different growth stages was designed, which is more in line with the growth pattern of hardhead trout and significantly improves the growth rate of hardhead trout.

[0016] Boosting immunity: Light of different wavelengths can effectively enhance the immunity of hardhead trout and reduce the incidence of disease.

[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, this invention provides a lighting method suitable for factory farming of hardhead trout at different growth stages. Based on the physiological characteristics of changes in opsin in the retina of hardhead trout at different growth stages, using ultraviolet, blue, and red light of different wavelengths at different growth stages can effectively improve the growth rate, immunity, and health level of hardhead trout. This method not only significantly improves farming efficiency but also has broad application prospects, especially in the large-scale industrial farming of hardhead trout, where it demonstrates significant economic benefits. Attached Figure Description

[0020] Figure 1 Schematic diagram of light regulation in factory farming of hardhead trout at different growth stages Detailed Implementation

[0021] The basic principle of this invention is to improve the growth and immune performance of hardhead trout by controlling the lighting method, as shown in the light intensity versus body weight curve. Figure 1 As shown.

[0022] 1: Illumination methods for stages smaller than 3 g When hardhead trout are less than 3 g in weight, they are irradiated with ultraviolet light with a wavelength of 360 nm-370 nm, with the light intensity controlled at 30 μmol / m² / s, for 12 hours daily. This method can significantly enhance the immunity of hardhead trout and reduce the incidence of diseases.

[0023] 2: Irradiation method for the 3 g-8 g stage When hardhead trout reached a weight of 3 to 8 g, they were irradiated with both ultraviolet (UV) and blue light. The UV light intensity was gradually reduced to 20 μmol / m² / s, while the blue light intensity was gradually increased from an 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 the hardhead trout improved simultaneously.

[0024] 3: Irradiation method for the 8 g-38 g stage During the stage when hardhead trout weighed more than 8 g but less than 38 g, ultraviolet light irradiation was discontinued, and blue light with a wavelength of 430 nm-445 nm was used for irradiation alone, with the light intensity controlled at 40 μmol / m² / s and the irradiation time being 12 hours per day. Blue light irradiation significantly promoted skeletal development and growth rate of hardhead trout during this stage.

[0025] 4: Irradiation methods for stages above 38 g When hardhead trout weigh more than 38 g, they are irradiated with red light with a wavelength of 560 nm-580 nm at an intensity of 50 μmol / m² / s for 12 hours per day. Red light at this stage promotes sexual maturity and gonadal development in hardhead trout, ensuring their reproductive health and overall growth quality, thus guaranteeing the continuation of aquaculture.

[0026] Implementation process

[0027] Based on the physiological characteristics of changes in opsin in the retina of hardhead trout at different growth stages, this invention employs different wavelengths of illumination schemes.

[0028] Research on fish rhodopsin has already been conducted. [4] We found that opsins in the retina of hardhead trout include RH1 in rod cells and SWS1, SWS2, RH2, and M / LWS in cone cells, which can sense light intensity, ultraviolet light, blue light, green light, and red light, respectively. As receptors for light environment response in fish, opsins influence physiological functions by regulating the central nervous system and endocrine system; therefore, a suitable light environment is of great significance for improving fish welfare.

[0029] Some research has also been conducted on the development of the retina in fish. [5]However, existing research is not comprehensive or systematic enough, especially regarding the expression of opsins and the spectral characteristics of visual pigments at different growth stages of hardhead trout, which require further refinement.

[0030] Current research on rhodopsin in hardhead trout mostly focuses on small-sized fish and lacks systematic research on the entire developmental stage. [6, 7] .

[0031] The expression of rod cells in fish varied significantly at different water depths, with a significantly higher expression level in deep-sea fish. [8] At different developmental stages of fish, the expression of opsin in cone cells varies significantly due to changes in the light environment in which they live. [5, 9, 10] .

[0032] Visual pigments are the core of the fish's visual system, determining the wavelength of maximum absorbance (λmax) to which fish are most sensitive. Fish exhibit significant differences in spectral adaptability at different developmental stages. Determining the optimal spectral range and peak value of hardhead trout based on the absorption spectra of visual pigments is more accurate, reliable, and better suited to their growth needs.

[0033] Existing studies on the effects of different spectra (light colors) on the physiology and biochemistry of rainbow trout have only limited the spectral wavelength range and have not set spectral peak values ​​based on the absorption spectral characteristics of fish visual pigments. This may be the reason for the inconsistency in the research results. [11-16] .

[0034] Therefore, this invention aims to address the above problems by exploring the morphological structure and distribution characteristics of opsins in the retina of hardhead trout at different growth stages from the following two aspects.

[0035] Research Content

[0036] (1) Characteristics of opsin gene expression in scleroderma at different life stages Real-time quantitative PCR (RT-qPCR) was used to determine the expression characteristics of five opsins in rod cells of the retina of hardhead trout during larval, juvenile, young, and adult stages, including RH1, SWS1, SWS2, RH2, and M / LWS, to explore the differences in light intensity and color requirements of hardhead trout at different life stages.

[0037] (2) Visual pigment absorption spectra of hardhead trout at different life stages

[0038] Microspectrophotometry (MSP) was used to determine the wavelengths of maximum absorbance (λ) of five opsins in the retinas of hardhead trout at larval, juvenile, and adult stages. maxThe study employed a visual photochromic pigment modeling method (MD) based on atomic and molecular dynamics simulations to validate and analyze the absorption spectrum range and peak value of the optimal photochromic pigment at different life stages of the hardhead trout.

[0039] Research Methods

[0040] (1) Characteristics of opsin gene expression in hardhead trout Real-time quantitative PCR (RT-qPCR) was used to determine the expression levels of various opsin genes in the retina of hardhead trout at the mRNA level. First, expression primers for the internal reference genes RH1, SWS1, SWS2, RH2, and M / LWS in hardhead trout were designed using Primer Premier 6.0 software. Amplification was performed using 18S rRNA, β-actin, ubiquitin, EF-1α, and β-tubulin as internal reference genes. The mRNA expression characteristics of RH1, SWS1, SWS2, RH2, and M / LWS opsin synthesis in the retina of hardhead trout at different life stages (larvae, juveniles, young fish, and adults) were compared.

[0041] (2) Visual pigment absorption spectrum of hardhead trout

[0042] The absorption spectra of visual pigments in hardhead trout were determined using a combination of microspectrophotometry (MSP) and visual pigment modeling based on atomic and molecular dynamics simulations (MD).

[0043] The method for determining the absorption spectra of visual pigments using MSP was based on Kondrashev (2022). Hardhead trout were acclimatized to darkness for 2-3 hours and then euthanized with 300 mg / L ethyl m-aminobenzoate methanesulfonate (MS-222, Sigma Chemicals Inc., USA). Eye tissue was then harvested under dark red light, and the retina was extracted and placed in a phosphate-buffered saline solution (PBS; Sigma-Aldrich) containing 6.0% sucrose. A portion of the retina was excised and placed on a glass slide, a suitable amount of PBS buffer was added, and the slide was covered with a coverslip and sealed with high-vacuum silicone grease. The wavelengths of maximum absorbance (λ) of each opsin were then measured using a microspectrophotometer. max ).

[0044] in conclusion

[0045] At a body weight of 3 g, the SWS1 protein content in the retina of hardhead trout was the highest, with a λmax between 360 nm and 370 nm. From 3 g to 8 g, the SWS1 protein content in the retina gradually decreased, while the SWS2 protein content gradually increased, with a λmax between 430 nm and 445 nm. The RH1 protein content also decreased. From 8 g to 38 g, the SWS1 protein disappeared from the retina, while the SWS2 protein gradually increased to its highest point. The RH1 protein content also gradually increased and was higher than that at a body weight of 3 g. When the body weight of hardhead trout exceeded 38 g, the SWS2 protein content decreased, while the M / LWS protein remained at a high level, with a λmax between 560 nm and 580 nm. The RH1 protein content continued to increase and then stabilized, remaining higher than that at a body weight of 8 g to 38 g.

[0046] By investigating the expression levels of opsin and the spectral characteristics of visual pigment absorption in the retina of hardhead trout at different developmental stages, this invention establishes a supplemental lighting strategy for hardhead trout at different growth stages.

[0047] 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 ofSchizochytrium sp. whole cell biomass by Atlantic salmon ( Salmo salar ), Aquaculture, 533, 736156. [4] Liu Chuwu et al., 2015. Research progress on fish opsins, Marine and Limnological Journal, 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 intensive farming of hardhead trout at different growth stages based on opsin, characterized by: Based on the expression characteristics of RH1 protein in rod cells and SWS1, SWS2, RH2, and M / LWS opsins in cone cells of the retina of hardhead trout, and the detection results of the maximum absorbance wavelength of visual pigments of the five opsins in the retina of hardhead trout, growth stages were divided according to body weight and corresponding illumination schemes were adopted, as follows: For larvae weighing less than 3 g, the SWS1 protein content in the retina is the highest, and the λmax is between 360 nm and 370 nm. Ultraviolet light with a wavelength of 360 nm to 370 nm is used for irradiation. During the juvenile stage of hardhead trout, when the weight reaches 3 g to 8 g, the content of SWS1 protein in the retina gradually decreases, while the content of SWS2 protein gradually increases. The λmax of SWS2 protein is between 430 nm and 445 nm, while the RH1 protein shows a decreasing trend. Ultraviolet light with a wavelength of 360 nm to 370 nm and blue light with a wavelength of 430 nm to 445 nm are used for irradiation. In the juvenile stage of hardhead trout with a weight greater than 8 g but less than 38 g, SWS1 protein disappeared in the retina, while SWS2 protein gradually increased to a maximum point, and RH1 protein gradually increased and was higher than the expression level of hardhead trout with a weight of 3 g. Ultraviolet light irradiation was stopped and replaced with blue light irradiation with a wavelength of 430 nm-445 nm. For adult fish weighing over 38 g, SWS2 protein decreased, while M / LWS protein remained high. The λmax of M / LWS protein was between 560 nm and 580 nm. RH1 protein continued to increase and then stabilized, and its expression level was higher than that of hardhead trout weighing 8 g to 38 g. Red light with a wavelength of 560 nm to 580 nm was used for irradiation. During the larval stage, the light intensity was controlled at 30 μmol / m² / s, and the daily irradiation time was 12 hours. During the juvenile stage, the intensity of ultraviolet light was reduced to 20 μmol / m² / s, and the intensity of blue light was gradually increased from 10 μmol / m² / s to 30 μmol / m² / s, with the irradiation time maintained at 12 hours / day. During the juvenile stage, the light intensity was controlled at 40 μmol / m² / s, and the daily irradiation time was 12 hours. During the adult stage, the light intensity is 50 μmol / m² / s, and the irradiation time is maintained at 12 hours / day.

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