Method for distinguishing between female and male in intermediate sea urchin

Abstract

The application discloses a method for distinguishing female and male of intermediate sea urchin, and belongs to the field of sea treasure seed production. According to the color and brightness difference of the peristome of the intermediate sea urchin, the standard color value and threshold value of the female and male sea urchin are determined, and the female and male of the intermediate sea urchin is determined. The distinguishing method can determine the female and male of the intermediate sea urchin in vivo, and the determination method is more economical, convenient, efficient and practical, so that the distinguishing method has important application value for sea urchin breeding industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of sea treasure seed production, and particularly relates to a method for distinguishing between female and male in intermediate sea urchin. BACKGROUND

[0002] Sea urchin is a spiny animal with high economic value. In recent years, the market demand for sea urchin is increasing, and the price of sea urchin is also increasing. The most popular sea urchin in the market is Strongylocentrotus intermedius, also known as intermediate sea urchin. Intermediate sea urchin was initially introduced from Japan by Dalian Ocean University, and is now widely bred and cultured in the coastal areas of northern China.

[0003] The female-to-male ratio of intermediate sea urchin is basically 1:1, but the number of sperm released by a male sea urchin can meet the fertilization needs of nearly 100 female sea urchins. Therefore, the number of female sea urchin required in seed production is much larger than that of male sea urchin. Since it is difficult to distinguish between female and male sea urchin before the release of reproductive cells, the number of male sea urchin is prepared according to twice the number of female sea urchin in terms of the purchase, storage, maturation and use of sea urchin seeds, which results in the waste of male sea urchin and increases the production cost.

[0004] Seed production requires the injection of 0.5M KCl into the sea urchin body to release reproductive cells, and then the sea urchin is placed on a platform or a flat plate with the genital pore facing up. After the genital pore exudes reproductive cells, the female sea urchin is identified according to the orange-red reproductive cells, and the male sea urchin is identified according to the white reproductive cells. After seed production, the sea urchin basically dies in a short period of time. Therefore, there is an urgent need for a method to distinguish between female and male sea urchin in intermediate sea urchin seed production.

[0005] Han Yalan combined DNA sequencing technology to perform correlation analysis on the whole genome of sea urchin, and identified a SNP site related to the gender of intermediate sea urchin. The specific molecular marker was used to determine the gender of intermediate sea urchin (Research on a gender-specific molecular marker for intermediate sea urchin and the gender differentiation mechanism[J]. Dalian Ocean University, 2022.). Chuguan Chu et al. observed and analyzed sea urchin by using tissue sectioning method and microsatellite marker analysis method, and preliminarily determined the sites and corresponding alleles that have a certain linkage relationship with the gender determination site of intermediate sea urchin (Microsatellite analysis of the gender traits of intermediate sea urchin[J]. 2009. 24(S1): 35-39.). However, the two studies are cumbersome for the determination of the gender of intermediate sea urchin, as they require the extraction of genetic material of intermediate sea urchin, the design of primers for PCR amplification, and the difference in allele frequency due to the limited number of samples and single sampling site in the test. Therefore, the linkage relationship and degree of linkage between the obtained markers and the gender determination site also need to be further verified. SUMMARY

[0006] In view of the problems in the prior art, the present application aims to provide a method for distinguishing between female and male intermediate sea urchins, wherein the peristome membrane of the intermediate sea urchin has a bright-dark difference and shows different colors such as reddish brown, milk brown, milk white, milk gray, milk yellow, gray green, milk blue, and gray blue. The standard color value and the threshold value of the female and male sea urchins are determined according to the color and brightness difference of the peristome membrane of the intermediate sea urchin. The female and male intermediate sea urchins are determined by using a color difference meter. The distinguishing method is more economical, convenient, efficient, and practical.

[0007] To achieve the above object, the present application adopts the following technical solution:

[0008] In the first aspect of the present application, a method for distinguishing between female and male intermediate sea urchins is provided, which comprises the following steps:

[0009] (1) The L, a, and b values of the peristome membrane of 200-300 female intermediate sea urchins are measured by using a color difference meter. The median values L1, a1, and b1 of the L, a, and b values of all the female intermediate sea urchins are taken as the standard color value. The female intermediate sea urchins with a small difference value from the standard color value are screened, and the number of the screened female intermediate sea urchins is 20% of the total number of the female sea urchins. The maximum difference values ΔL1, Δa1, and Δb1 of the 20% of the screened female intermediate sea urchins from the standard color value are selected, and ΔE♀ is calculated as the threshold value for determining the female intermediate sea urchins.

[0010] The standard color value and the threshold value of the male intermediate sea urchins are screened and calculated in the same way as the female intermediate sea urchins. Specifically, the L, a, and b values of the peristome membrane of 200-300 male intermediate sea urchins are measured by using a color difference meter. The median values L2, a2, and b2 of the L, a, and b values of all the male intermediate sea urchins are taken as the standard color value. The male intermediate sea urchins with a small difference value from the standard color value are screened, and the number of the screened male intermediate sea urchins is 20% of the total number of the male sea urchins. The maximum difference values ΔL2, Δa2, and Δb2 of the 20% of the screened male intermediate sea urchins from the standard color value are selected, and ΔE♂ is calculated as the threshold value for determining the male intermediate sea urchins.

[0011] (2) The L1, a1, and b1 standard color value and the threshold value ΔE♀ of the female intermediate sea urchins determined in step (1) are taken as the determination standard, and are input into the color difference meter. If the measured ΔE of the sea urchin sample is less than ΔE♀, the color difference meter shows that it is qualified, and the sea urchin is determined to be female.

[0012] The L2, a2, and b2 standard color value and the threshold value ΔE♂ of the male intermediate sea urchins determined in step (1) are taken as the determination standard, and are input into the color difference meter. If the measured ΔE of the sea urchin sample is less than ΔE♂, the color difference meter shows that it is qualified, and the sea urchin is determined to be male.

[0013] The L value is the brightness of the peristomial membrane of the Strongylocentrotus intermedius, from 0-100, the larger the L value indicates the whiter (brighter), the smaller the L value indicates the darker (darker); the a value is the red-green value of the peristomial membrane of the Strongylocentrotus intermedius, which has positive and negative, +a represents red, -a represents green (not enough red); the b value is the yellow-blue value of the peristomial membrane of the Strongylocentrotus intermedius, which has positive and negative, +b represents yellow, -b represents blue.

[0014] The ΔE♀=(ΔL1 2 +Δa1 2 +Δb1 2 ) 1 / 2 , the ΔE♂=(ΔL2 2 +Δa2 2 +Δb2 2 ) 1 / 2 , the ΔE=(ΔL3 2 +Δa3 2 +Δb3 2 ) 1 / 2 .

[0015] The ΔL3 is the difference between the L value of the measured sea urchin gonad sample and the L standard color value, the Δa3 is the difference between the a value of the measured sea urchin gonad sample and the a standard color value, and the Δb3 is the difference between the b value of the measured sea urchin gonad sample and the b standard color value.

[0016] The measured diameter of the Strongylocentrotus intermedius is 4.5-7.0 cm.

[0017] When measuring the L, a, and b values of the peristomial membrane of the Strongylocentrotus intermedius, the ambient light is below 200 lx.

[0018] The temperature during measurement is 5-22℃.

[0019] Before the measurement operation, a black pad with a thickness of 1 cm and an inner diameter consistent with the aperture (4 cm) of the positioning plate (4 cm) that comes with the color difference meter (4 cm) is made of rubber products, the positioning plate attached to the color difference meter is pasted onto the black pad, and the inner diameter of the pad matches the inner diameter of the positioning plate. Figure 1 Figure 3 The measurement time of the L, a, and b values of the Strongylocentrotus intermedius is within 1-3 minutes after the sea urchin is taken out of the water. Figure 4 Figure 5 During the measurement operation, the sea urchin mouth does not move.

[0020] During the measurement operation, the sea urchin mouth does not move.

[0021] During the measurement operation, the sea urchin mouth does not move.

[0022] ​​In a second aspect, the application provides the use of the above method in the determination of the sex of the juvenile sea urchin.

[0023] Advantages of the application:

[0024] The application establishes a method for distinguishing the sex of juvenile sea urchins by using a color difference meter to detect the color and brightness differences of the peristomial membrane of juvenile sea urchins, which can determine the sex of juvenile sea urchins in vivo, and the determination method is more economical, convenient, efficient, and practical, thus having important application value for sea urchin aquaculture. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Photo of color difference meter standing upright

[0026] Figure 2 Photo of color difference meter lying down

[0027] Figure 3 Photo of positioning plate attached to color difference meter

[0028] Figure 4 Photo of black pad

[0029] Figure 5 Photo of black pad and positioning plate pasted together

[0030] Figure 6 Photo of juvenile sea urchin and peristomial membrane

[0031] Figure 7 Photo of using color difference meter to determine female sea urchin (unqualified)

[0032] Figure 8 Photo of using color difference meter to determine female sea urchin (qualified)

[0033] Figure 9 Photo of using color difference meter to determine male sea urchin (unqualified)

[0034] Figure 10 Photo of using color difference meter to determine male sea urchin (qualified) DETAILED DESCRIPTION

[0035] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0036] As described above, it is difficult to distinguish the male and female of the intermediate sea urchin before the intermediate sea urchin releases the reproductive cells under the action of the labor induction, and the intermediate sea urchin will die in a short time after the labor induction, and the male sea urchin needs to be prepared according to 2 times of the number of the female sea urchin in the intermediate sea urchin breeding, which increases the production cost.

[0037] Based on this, the present application provides a simple and economical method for distinguishing the male and female of the intermediate sea urchin (the shell diameter is 4.5-7.0 cm) by the difference of the color and brightness of the peristome of the intermediate sea urchin, which has important significance for the breeding production of the intermediate sea urchin.

[0038] When the color difference instrument is used for detection, the sea urchin needs to be in a state that the mouth tooth is not protruding and the spines are not swinging, so as to avoid that the mouth tooth or the spines swing and stretch above the peristome due to the protrusion of the mouth tooth, and affect the final measurement result.

[0039] Before measurement, a rubber product with a thickness of 1 cm and an inner diameter consistent with the hole diameter of the positioning plate attached with the color difference instrument is used to make a pad, the positioning plate attached with the color difference instrument is pasted on the pad, when measurement is performed, the pad is placed above the peristome of the sea urchin, and the center of the hole of the positioning plate is opposite to the center of the mouth tooth of the sea urchin. Then the measurement head of the color difference instrument is placed in the hole of the positioning plate, the switch of the color difference instrument is started, the light beam is emitted from the small hole of the measurement head, and the measurement point is irradiated to perform measurement. The added pad can avoid that the measurement head is placed on the spines of the sea urchin, and cause detection error.

[0040] The specific embodiments of the present application are further described in detail below with reference to the examples. The following detailed description is illustrative only and is not intended to limit the scope of the present application.

[0041] Example 1: Calculation of standard color value and threshold value when the male and female of the intermediate sea urchin are distinguished

[0042] In early October, the sea urchins with a shell diameter of 4.5-7.0 cm were taken from the sea urchin pool in the sea urchin breeding room under the light of 100 lx and the air temperature of 18℃, the positioning plate (with a black hollow pad) was placed above the peristome of the sea urchin, and then the L, a and b values of the peristome were measured by using the color difference instrument (LS173, Shenzhen Linshang Technology Co., Ltd., No. 1, Xin'an 2nd Road, Wuxi District, Baoan District, Shenzhen, Guangdong Province). Figure 5

[0043] A total of 445 sea urchins were measured, and 442 sea urchins released the reproductive cells after the 0.5M Kcl labor induction, of which 231 were female and 211 were male.

[0044] ​231 The median values of L, a, b of the peristomial membrane of the female sea urchin are: L1 = 13.77, a1 = 1.52, b1 = 3.32. Among the 46 sea urchins with L values closest to the L1 value 13.77, the largest absolute value of ΔL is 1.59; among the 46 sea urchins with a values closest to the a1 value 1.52, the largest absolute value of Δa is 0.57; among the 46 sea urchins with b values closest to the b1 value 3.32, the largest absolute value of Δb is 0.89; ΔE♀ = (ΔL1 + Δa1 + Δb1) = (2.5281 + 0.3249 + 0.7921) = 1.91. 2 2 2 1 / 2 1 / 2

[0045] 211 The median values of L, a, b of the peristomial membrane of the male sea urchin are: L2 = 15.83, a2 = 1.54, b2 = 4.80. Among the 42 sea urchins with L values closest to the L2 value 15.83, the largest absolute value of ΔL is 1.52; among the 42 sea urchins with a values closest to the a2 value 1.54, the largest absolute value of Δa is 0.61; among the 42 sea urchins with b values closest to the b2 value 4.80, the largest absolute value of Δb is 0.79; ΔE♂ = (ΔL2 + Δa2 + Δb2) = (2.31 + 0.37 + 0.62) = 1.82. 2 2 2 1 / 2 1 / 2

[0046] Example 2: Accuracy rate detection of the determination of the female and male of the intermediate sea urchin using the method established in the present application

[0047] Using the Lab mode, the standard L1, a1, b1 values and the threshold ΔE♀ of the female sea urchin are input into the color difference meter, and 300 sea urchin eggs are measured. The sea urchins that are qualified by the color difference meter are female sea urchins, and the sea urchins that are unqualified are discarded. As a result, 33 female sea urchins are screened out. The 33 sea urchins are induced to spawn and dissected, and it is confirmed that 29 are female and 4 are male. The accuracy rate of the method provided in the present application for detecting female sea urchins is 87.9%.

[0048]

[0049]

[0050] ​​​​​​​​​​The standard L2, a2, b2 values and threshold value ΔE♂ of male sea urchins are input into the color difference meter in Lab mode, 267 cultured seed urchins (of which 33 are determined to be female and have been removed) are measured, and the color difference meter presents qualified male sea urchins and unqualified ones are discarded. As a result, 36 male urchins are screened out. The 36 sea urchins are induced to spawn and dissected, and it is confirmed that 31 are male and 5 are female. The accuracy of the method provided in the present application for detecting male sea urchins is 86.1%.

[0051]

[0052]

[0053] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made by those skilled in the art based on the spirit and principles of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for sexing intermediate sea urchins, characterized by, It comprises the following steps: (1) The L, a, b values of 200-300 female intermediate sea urchin gonochore membrane parts are measured by using a color difference meter. The median values L1, a1, b1 of all the measured L, a, b values of the female intermediate sea urchin gonochore membrane parts are used as standard color values. The female intermediate sea urchins with smaller difference values from the standard color values are screened, and the number of the screened female intermediate sea urchins is 20% of the total number of the female sea urchins. The maximum ΔL1, Δa1, Δb1 of the 20% screened female intermediate sea urchins from the standard color values are selected, and ΔE♀ is calculated as the threshold value for determining the female intermediate sea urchins. The L, a, b values of 200-300 male intermediate sea urchin gonochore membrane parts are measured by using a color difference meter. The L2, a2, b2 standard color values and the threshold value ΔE♂ of the male intermediate sea urchin gonochore are obtained by the same method; (2) The L1, a1, b1 standard color values and the threshold value ΔE♀ of the female intermediate sea urchin gonochore measured in step (1) are used as the determination criteria, and are input into the color difference meter. If the measured sea urchin gonochore sample ΔE is less than ΔE♀, the color difference meter shows that it is qualified, and it is determined as a female gonochore. If the measured sea urchin gonochore sample ΔE is greater than ΔE♀, the color difference meter shows that it is unqualified, and it is not determined as a female or male; The L2, a2, b2 standard color values and the threshold value ΔE♂ of the male intermediate sea urchin gonochore measured in step (1) are used as the determination criteria, and are input into the color difference meter. If the measured sea urchin gonochore sample ΔE is less than ΔE♂, the color difference meter shows that it is qualified, and it is determined as a male gonochore. If the measured sea urchin gonochore sample ΔE is greater than ΔE♂, the color difference meter shows that it is unqualified, and it is not determined as a female or male; said ΔE♀ = (ΔL1 2 + Δa1 2 + Δb1 2 ) 1 / 2 , said ΔE♂ = (ΔL2 2 + Δa2 2 + Δb2 2 ) 1 / 2 , said ΔE = (ΔL3 2 + Δa3 2 + Δb3 2 ) 1 / 2 ; The measured intermediate sea urchin shell diameter is 4.5-7.0 cm; When the color difference meter is used to measure the L, a, b values of the intermediate sea urchin gonochore membrane parts, a gasket plate with the same diameter as the positioning plate hole diameter provided with the color difference meter is first prepared. The positioning plate provided with the color difference meter is pasted onto the gasket plate. The inner diameter of the gasket plate is consistent with the inner diameter of the positioning plate. When measuring, the gasket plate is placed above the sea urchin gonochore membrane, and the center of the positioning plate hole is opposite to the center of the sea urchin mouth. The environmental light for measuring the L, a, b values of the intermediate sea urchin gonochore membrane parts is below 200 lx. The temperature during the measurement is 5-22℃. The measurement time of the L, a, b values of the intermediate sea urchin is within 1-3 minutes after the sea urchin is taken out of the water, and the measurement is performed when the sea urchin mouth is not active.

2. The method of claim 1 is applied in the determination of the female and male intermediate sea urchin gonochore.

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