A method for evaluating abalone disease resistance based on infrared heartbeat detection

Abstract

The present invention is a method for evaluating the disease resistance of abalone based on infrared heartbeat detection, and the steps include: laboratory taming of abalone to be detected; connecting an abalone heartbeat detection device and turning it on, pasting an infrared probe on the shell surface near the heart position of abalone, obtaining the abalone heartbeat signal through an infrared probe, amplifying it with an AMP03 amplifier, and collecting it with a Powerlab signal receiver, and then importing it into a computer for analysis; injecting abalone-derived pathogenic bacteria from the upper foot position of the abalone to be tested; using LabChart software to continuously record the abalone heartbeat signal in real time during the pathogen stress process, and statistically analyzing the heart rate after the acquisition; according to the heartbeat law of abalone during the stress process, developing two indicators of the pathogen stress heart rate inflection point CBTB and the pathogen stress heart rate zero point CZTB, and evaluating the disease resistance of abalone according to the CBTB and CZTB numerical values. The evaluation method of the present invention has the advantages of real-time, accuracy, intuitiveness, quantification, etc., can realize the synchronous evaluation of a large number of experimental abalone, and can be applied to the comparative study of pathogen tolerance between different groups or genotypes of abalone.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a method for evaluating abalone disease resistance based on infrared heartbeat detection. Background Art

[0002] Abalone, belonging to the phylum Mollusca, class Gastropoda, subclass Prosobranchia, order Archaeo gastropoda, family Haliotididae, has tender meat and delicious taste. It is a seafood with high nutritional value. In the 1970s, artificial breeding of Abalone variegata and Abalone discus humpback was achieved, and factory breeding was gradually carried out. In the early 1990s, Fujian, Guangdong and other southern coastal provinces established land-based factory breeding technology of Abalone variegata through introduction and absorption, and since then, the large-scale abalone breeding industry has begun to develop. In 2000, Abalone discus humpback, which was originally cultivated in Liaoning Province, was introduced to Fujian. Combined with the marine environment of Fujian, a large-scale shallow sea raft breeding model was gradually established, which led to an explosive growth in the breeding scale and output of Abalone discus humpback, and gradually became the dominant abalone breeding species in my country. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), my country's abalone farming output reached 180,300 tons in 2019, accounting for more than 90% of the world's total abalone farming output, of which Fujian Province's abalone farming output was 143,000 tons, accounting for 79.3% of the country's abalone farming output. Abalone has become the aquaculture species with the highest output value in Fujian Province due to its high economic benefits. The development and growth of the abalone farming industry has driven the rapid development of abalone farming equipment, seaweed farming, compound feed, processing and transportation industries. A complete industrial chain has now been formed, creating huge economic, social and ecological benefits. At present, artificial breeding of abalone in my country is mainly concentrated in Fujian, Shandong and Guangdong. The wrinkled disc abalone (Haliotis discus hannai) is currently the main economic abalone species in my country, while the variegated abalone (Haliotis diversicolor) is partially farmed in some southern provinces.

[0003] Diseases have always been one of the unfavorable factors affecting the yield of abalone aquaculture. As an aquatic animal, abalone has a strong material exchange between its endocrine environment and the external water environment, so it is easily stressed by harmful microorganisms in the environment. Diseases will affect the growth, development, reproduction, metabolism, behavior and other physiological activities of abalone. Immunity to harmful microorganisms is considered to be an important trait of economic shellfish. It determines the survival rate of farmed shellfish during the farming process to a certain extent, thus directly affecting its final yield. Based on the needs of industry and theory, it is necessary to establish a method for measuring the immune tolerance of marine shellfish. In the early days, it was mostly assessed by subjecting a certain shellfish to acute or chronic pathogen stress and measuring its survival rate or half-lethal concentration (50% Lethal concentration, LC50). This method can more intuitively reflect the immune ability of shellfish, but this method also has certain defects. Its determination process is relatively long (generally at least 96 hours), and a large number of experimental animals need to be sacrificed during the determination process, and the physiological mechanism of shellfish immune activity cannot be known. Later, researchers combined other physiological measurement methods such as cellular immune parameter measurement (blood cell number, blood cell phagocytic activity, blood cell respiratory burst capacity) and humoral immune parameter measurement (immune-related enzyme activity) to comprehensively evaluate and explain the immune tolerance characteristics of marine shellfish. However, these methods can only indirectly and partially reflect the immune capacity of shellfish, and lack intuitiveness and sensitivity. Therefore, a convenient, reliable and intuitive method is needed to measure the immune tolerance of marine shellfish species.

[0004] Using infrared principle to detect the heartbeat of shellfish has always been a simple, fast and highly accurate technical means to reflect the changes in the physiological state of shellfish. As early as 1990, researchers successfully used infrared detection methods to observe the stable heartbeat of several crustaceans and shellfish. Later, researchers used heartbeat detection to study the effect of temperature on the physiological state of shellfish. Stillman et al. used Arrhenius break temperatures (ABT) to reflect the tolerance of porcelain crabs to high temperatures. They found that within a certain temperature range, the heart rate of crabs increased with the increase of temperature; after exceeding a certain temperature point, the heart rate of crabs decreased with the increase of temperature. This inflection point temperature is ABT, which represents the extreme value of invertebrate tolerance to high temperature. When the temperature exceeds this point, the metabolic function of organisms decreases or becomes unbalanced, which is manifested as a decrease in heart rate. The value of this ABT can be obtained by calculating the intersection of two linear Arrhenius equations before and after ABT. ABT has been widely used to evaluate the high temperature tolerance of shellfish species such as abalone, scallops, shrimps, snails, limpets, mussels, etc. Later, researchers successfully used the heartbeat index to evaluate the response of invertebrates such as abalone, scallops, and crayfish to adverse environmental factors such as low temperature, hypoxia, dew, and acidification. Based on this, they developed indicators such as abalone breakpoint dissolved oxygen (BPDO) and scallop heartbeat resistance comprehensive index (Yi) that can indicate the resistance of shellfish to specific environmental factors.

[0005] However, there is no research on the heartbeat change pattern of abalone under pathogen stress. Elucidating the changes in heartbeat function of abalone under pathogen stress and developing an accurate, reliable and quantifiable pathogen tolerance heartbeat index based on this will help us understand the heartbeat change patterns and characteristics of marine invertebrates under pathogen stress, which can be used to compare pathogen tolerance between different groups or genotypes of abalone, and can also be used as a breeding index in the genetic breeding of abalone disease resistance. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for evaluating the disease resistance of abalone based on infrared heartbeat detection. The method uses a Powerlab heartbeat detection system to detect and record the heartbeat waveform of abalone after injection of pathogens in real time, and calculates and analyzes the heartbeat frequency of abalone through LabChart software. Based on the changes in the heartbeat waveform and heart rate frequency of abalone under pathogen stress, an indicator of the immune heartbeat of abalone is developed to intuitively display the changing pattern of the heartbeat of abalone under pathogen stress, which is also conducive to comparing the immune characteristics between different abalone groups or genotypes. The proposal of the present invention helps to solve the problem of the lack of a real-time, accurate, intuitive and quantifiable disease resistance evaluation method for abalone (and other marine invertebrates).

[0007] A method for evaluating abalone disease resistance based on infrared heartbeat detection comprises the following steps:

[0008] Step 1: The abalone to be tested is domesticated under controlled acclimation conditions indoors for 7-14 days to adapt it to the laboratory environment;

[0009] Step 2: Connect the abalone heartbeat detection device before the test;

[0010] Step 3, clean the attachments on the surface of the abalone shell and wipe off the moisture; turn on the Powerlab signal receiver of the abalone heartbeat detection device and the power of the computer, set the LabChart software in the computer to the signal acquisition state, press the infrared probe of the abalone heartbeat detection device against the abalone shell surface at the abalone heart position, observe the heartbeat waveform state detected in the LabChart software at the same time, and fine-tune the position of the infrared probe to make the heartbeat waveform regular and clear; then use strong adhesive glue to stick the infrared probe to the corresponding shell surface position;

[0011] Step 4: Place the abalone with the probe attached into a temporary storage tank and store it at 20-25°C for 2-4 hours to adapt it to the detection environment;

[0012] Step 5: inject 50-100uL of abalone pathogenic bacteria from the upper foot of the experimental abalone;

[0013] Step 6: During the pathogen stress process, the abalone heartbeat signal was recorded in real time using LabChart software until the heartbeats of all experimental abalones stopped beating normally, and the recording was terminated. After the collection was completed, the abalone heart rate was statistically analyzed;

[0014] Step seven, according to the heartbeat law of abalone during the process of pathogen stress, define the inflection point time when the abalone heart rate begins to decrease minus the pathogen injection time as the abalone pathogen stress heart rate inflection point, and the time when the abalone heart rate drops to zero is the pathogen stress heart rate zero point; combine the two indicators CBTB and CZTB to comprehensively reflect the tolerance of abalone heartbeat function under pathogen stress, so as to evaluate the disease resistance of abalone; the larger the CBTB value, the stronger the adaptability of the abalone heartbeat to pathogens; the larger the CZTB value, the stronger the ability of abalone to resist the decline in heart rate under pathogen stress.

[0015] Preferably, in step 1, the shell length of the abalone to be tested is not less than 2.0 cm; the acclimation conditions include: temperature 20-25°C, salinity 30-33psu, pH 7.8-8.2, dissolved oxygen ≥5.0mg / L, continuous aeration of the aquaculture water, full water replacement every day, feeding fresh Gracilaria, kelp or laver during the acclimation period and timely cleaning of residual bait, feces, pseudo-feces, and dead abalone.

[0016] Preferably, the abalone heartbeat detection device in step 2 includes: a temporary holding tank, an insulation tank, a Powerlab signal receiver, an AMP03 signal amplifier, an infrared probe, a temperature-controlled circulating water bath, a temperature recorder, a computer, and LabChart software; the temporary holding tank is placed in the insulation tank, and the seawater in the insulation tank is connected to the temperature-controlled circulating water bath through a water pipe, and the water temperature in the temporary holding tank is monitored and recorded by a temperature recorder to control the temperature of the seawater in the temporary holding tank; the infrared probe is fixed to the shell surface at the position of the abalone heart, and then connected to the AMP03 signal amplifier through an RJ11 connection, and the AMP03 signal amplifier is connected to the Powerlab signal receiver in a BNC manner to capture, amplify and collect the abalone heartbeat signal; the Powerlab signal receiver is connected to the computer via USB, and the collected heartbeat signal is visualized and the heart rate is analyzed through LabChart software.

[0017] Preferably, in step three, the attachments are attached organisms, dirt, mucous membranes, etc.; the strong adhesive glue is 502 glue or Blu-Tag blue tack glue.

[0018] Preferably, in step five, the injection concentration of the abalone-derived pathogenic bacteria is the 96h minimum 100% lethal concentration of the experimental abalone population.

[0019] Preferably, in step seven, the heartbeat pattern of abalone during the pathogen stress process is divided into two stages according to its characteristics: a. normal heartbeat stage: the heartbeat waveform of abalone is normal and the heartbeat frequency is stable; b. abnormal heartbeat stage: the heartbeat waveform of abalone begins to become abnormal, manifested as irregular waveform, occasional cardiac arrest, and a reduced heart rate.

[0020] The advantages and technical effects of the present invention are:

[0021] The present invention discloses a method for evaluating abalone disease resistance based on infrared heartbeat detection. It describes the heartbeat and heart rate characteristics of abalone under pathogen stress in an infrared heartbeat detection mode, and develops two abalone disease resistance heartbeat indicators, namely, the heart rate turning point CBTB under pathogen stress and the heart rate zeroing point CZTB under pathogen stress, based on these characteristics. These indicators can be used to evaluate the adaptability of abalone to pathogen stress and the ability of abalone to resist the decrease in heart rate under pathogen stress, thereby comprehensively indicating the immune tolerance of abalone. The evaluation method of the present invention has the advantages of real-time, accuracy, intuition, and quantification. It can also realize the synchronous evaluation of large batches of experimental animals by matching the Powerlab host with a high-throughput channel. The obtained data can help us understand the heartbeat change rules and characteristics of marine invertebrates under pathogen stress, which can be applied to the comparison of immune tolerance between different groups or genotypes of abalone, and can also be used as a breeding indicator in the genetic breeding of abalone disease resistance. The method of the present invention is the first to use heartbeat detection to evaluate the immune tolerance of abalone. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the abalone heartbeat detection device in the present invention; (the thick solid line in the figure represents the outer wall of the instrument / device; the thin solid line represents the connecting line; the double solid line represents the water pipe; the arrow represents the direction of water flow; the dotted line represents the water level);

[0023] Figure 2 The heartbeat waveform diagram of abalone in the process of pathogen stress in the present invention; (in the figure, (A) is a normal heartbeat waveform, (B) is an abnormal heartbeat waveform, and the arrow indicates an abnormal heartbeat);

[0024] Figure 3 Schematic diagram of heart rate change during abalone pathogen stress in the present invention; (in the figure, the linear equation of abalone heart rate and exposure time is marked above the fitting line; intersection 1 represents the abscissa corresponding to the intersection of the two linear equations; intersection 2 represents the abscissa when the ordinate of the linear equation in the abnormal heart rate stage is 0);

[0025] Figure 4 The figure is a schematic diagram for comparing the CBTB and CZTB values ​​of the three genotypes of Haliotis discus, Abalone occidentalis and Haliotis discus in the present invention; in the figure, lowercase letters (a, b) indicate significant differences in CBTB between different genotypes (p < 0.05); uppercase letters (A, B) indicate significant differences in CZTB between different genotypes (p < 0.05). DETAILED DESCRIPTION

[0026] In order to further understand the content, features and effects of the present invention, the following embodiments are given as examples and described in detail with reference to the accompanying drawings. It should be noted that the embodiments are illustrative, not restrictive, and the protection scope of the present invention cannot be limited thereby.

[0027] The present invention provides a method for evaluating abalone disease resistance based on infrared heartbeat detection, and the specific implementation steps are as follows:

[0028] 1) Laboratory acclimatization of abalone to be tested: The abalone to be tested is acclimatized for 7-14 days under suitable and controllable laboratory conditions to adapt it to the laboratory environment;

[0029] 2) Connection of the heartbeat detection device: Connect the components of the abalone heartbeat detection device, including the temporary holding tank, the heat preservation tank, the Power lab signal receiver, the AMP03 signal amplifier, the infrared probe, the temperature-controlled circulating water bath, the temperature recorder and the computer;

[0030] 3) Pasting of infrared probe for heartbeat detection: clean the attachments on the surface of the experimental abalone shell and wipe off the moisture. Turn on the power of the Powerlab signal receiver and the computer, set the LabChart software to the signal acquisition state, press the infrared probe against the surface of the abalone shell at the heart position of the abalone, observe the heartbeat waveform state detected in LabChart, and fine-tune the position of the infrared probe to make the heartbeat waveform regular and clear. Then use strong adhesive glue to paste the infrared probe to the corresponding shell surface position;

[0031] 4) Temporary culture before testing of experimental abalone: ​​Put the abalone with the infrared probe attached into a temporary culture tank pre-filled with seawater, which is placed in an insulation tank pre-filled with seawater, which is connected to a temperature-controlled circulating water bath via a water pipe. By adjusting the temperature of the water bath, the water temperature in the temporary culture tank is controlled at 20-25°C. The experimental abalone is temporarily cultured at 20-25°C for 120-240 minutes to fully adapt to the testing environment;

[0032] 5) Injection of abalone pathogenic bacteria into the foot muscle: Use a sterile microinjector to inject 50-100uL of abalone pathogenic bacteria into the experimental abalone. The injection concentration is the minimum 100% lethal concentration of the experimental abalone population within 96 hours;

[0033] 6) Heartbeat data collection and analysis: During the temporary culture and pathogen stress process, the abalone heartbeat signal was recorded in real time using LabChart software until the heartbeats of all experimental abalone stopped beating normally. After the collection was completed, the heart rate was statistically analyzed;

[0034] 7) Analysis of heartbeat patterns and development of indicators: The heartbeat of abalone during pathogen stress can be divided into two stages according to its characteristics: normal heartbeat stage and abnormal heartbeat stage. The linear fitting equations of the abalone heart rate change over time in the two stages were established respectively. The horizontal coordinate time at the intersection of the two equations was taken, and the time when the bacteria were injected was subtracted to obtain the heart rate inflection point (cardiac breaktimepoint upon bacteria, CBTB, in min) of abalone pathogen stress; according to the linear equation of the abnormal heartbeat stage, the time when the heart rate was 0 was calculated, and the heart rate zero point (cardiaczero timepoint upon bacteria, CBZB, in min) of pathogen stress was subtracted from the above CBTB time. Combining the two abalone pathogen tolerance indicators CBTB and CZTB, the adaptability of abalone to pathogen stress and the ability of abalone to resist the decrease in heart rate under pathogen stress can be evaluated, thereby comprehensively evaluating the disease resistance of abalone.

[0035] In order to more clearly illustrate the specific implementation mode of the present invention, an embodiment is provided below:

[0036] The present invention discloses a method for evaluating abalone immune tolerance based on infrared heartbeat detection, which has the advantages of real-time, accuracy and high efficiency, and improves the traditional method based on pathogen stress survival rate or half-lethal concentration (LC50). 50 ) The shortcomings of the method for evaluating the immune tolerance of marine invertebrates. By quantitatively describing the changing trends of abalone heartbeat and heart rate under pathogenic stress, two abalone immune heartbeat indicators have been developed, which can indicate the adaptability of abalone heart function under pathogenic stress and the ability to resist heart rate drop. The present invention is achieved through seven specific steps: laboratory taming of abalone to be tested, connection of a heartbeat detection device, pasting of a heartbeat detection infrared probe, temporary culturing of experimental abalone for testing, injection of abalone pathogenic bacteria into the foot muscle, heartbeat data collection and analysis, heart rate law analysis and indicator development. The specific implementation methods of the present invention will be further described below in conjunction with the accompanying drawings.

[0037] 1) Laboratory domestication of abalone to be tested

[0038] Abalone with regular appearance and good vitality were selected for the heartbeat detection experiment. The size of abalone was ≥2.0cm. Before the experiment, abalone was domesticated under controlled conditions indoors for 7-14 days to adapt them to laboratory conditions. Domestication conditions include: temperature 20-25℃, salinity 30-33psu, pH 7.8-8.2, dissolved oxygen ≥5.0mg / L, continuous aeration of the aquaculture water, and full replacement of domestication seawater every day. Other domestication seawater indicators should meet the requirements of the Fishery Water Quality Standard (GB 11607-89). During the domestication period, fresh Gracilaria, kelp or laver were fed, and residual bait, feces, pseudo-feces, dead abalone individuals, etc. were cleaned up in time.

[0039] 2) Heartbeat detection device connection

[0040] As attached Figure 1 As shown in the figure, the abalone heartbeat detection device is mainly composed of a temporary holding tank, a heat preservation tank, a Powerlab signal receiver, an AMP03 signal amplifier, an infrared probe, a temperature-controlled circulating water bath, a temperature recorder, and a computer. Each part is connected in the following way:

[0041] a. The temporary tank is placed in a heat preservation tank. The seawater in the heat preservation tank is connected to a temperature-controlled circulating water bath through a water pipe to control the temperature of the seawater in the temporary tank. The water temperature in the temporary tank is monitored and recorded in real time through a temperature recorder;

[0042] b. The infrared probe is fixed to the shell surface at the heart position of the abalone by super glue, and then connected to the AMP03 signal amplifier via RJ11 connection, which is then connected to the Powerlab signal receiver via BNC to achieve the capture, amplification and collection of heartbeat signals;

[0043] c. The Powerlab signal receiver is connected to a computer (with LabChart software installed) via USB, and the collected heartbeat signals are visualized and analyzed using LabChart software.

[0044] 3) Paste the heartbeat detection infrared probe

[0045] a. Turn on the power of the Powerlab signal receiver and the computer, and set the LabChart software to the data acquisition state;

[0046] b. Place the abalone to be tested in a clean tray, clean the surface of the abalone shell, remove excess attached organisms, dirt, mucous membranes, etc., and then wipe the moisture on the shell surface with gauze. Press the infrared probe against the abalone shell surface at the heart position of the abalone, observe the heartbeat waveform state detected by the corresponding channel in LabChart at the same time, and fine-tune the position of the infrared probe to make the heartbeat waveform regular and clear. Then use hydrophobic strong adhesive glue (502 glue or Blu-Tag blue tack glue) to stick the infrared probe to the adjusted shell surface position, and wait for 1-2 minutes for the glue to completely solidify;

[0047] 4) Temporary storage before testing of experimental abalone

[0048] Place the abalone with the infrared probe affixed to it into a temporary holding tank (pre-installed with seawater at a temperature of 20-25°C, 23°C in this case). The abalone temporary holding tank is placed in an insulation tank (pre-installed with seawater). The insulation tank is connected to a temperature-controlled circulating water bath via a water pipe. A temperature recorder is used to detect and record the temperature in the temporary holding tank in real time. By controlling the temperature of the circulating water bath, the temperature of the abalone is controlled at 20-25°C (23°C in this case) during temporary holding. Temporary holding lasts for 120-240 minutes (200 minutes in this case) until the abalone fully adapts to the testing environment;

[0049] 5) Injection of abalone pathogenic bacteria into the foot muscle

[0050] Use a sterile microinjector to inject pathogenic bacteria into the abalone to be tested. The pathogen used is abalone pathogenic bacteria (in this case, abalone Vibrio harveyi), and the injection method is upper foot muscle injection, the injection volume is 50-100uL (50uL in this case), and the injection concentration is the minimum 100% lethal concentration of the experimental abalone in 96 hours (determined by preliminary experiments, in this case, 2×10 9 cfu / mL). At the same time, the same number of abalone to be tested was randomly selected and injected with an equal volume (50uL in this example) of sterile seawater as a control group.

[0051] 6) Heartbeat data collection and analysis

[0052] During the temporary culture and pathogen stress process, the abalone heartbeat signal was recorded in real time using LabChart software until the heartbeats of all experimental groups of abalone stopped beating normally. After the experiment, the built-in data analysis program of LabChart was used to identify the heartbeat of abalone and count the heart rate, and the heart rate value of abalone during the exposure period was calculated in beats / min. -1 (BPM);

[0053] 7) Heartbeat pattern analysis and indicator development

[0054] The heartbeat of abalone during the process of pathogen stress can be divided into two stages according to its characteristics: normal heartbeat stage and abnormal heartbeat stage. In the normal heartbeat stage, the heartbeat waveform of abalone is relatively normal, showing a main peak plus a secondary peak (see Appendix Figure 2 A), and the heart rate is relatively stable; in the abnormal heartbeat stage, Bao's heartbeat waveform presents an irregular multi-peak shape, with intermittent cardiac arrest and a reduced heart rate (Appendix Figure 2 B). Based on the above-mentioned changes in abalone heartbeat under pathogenic bacteria stress, two evaluation indicators for indicating abalone pathogen tolerance were developed. The specific methods are as follows:

[0055] a. Normal heartbeat stage: Bao's heart rate changes linearly (increases or decreases, Figure 3 ). Establish a linear fitting equation between the heart rate and exposure time of this stage (in this case, y = 0.0053x + 53.37);

[0056] b. Abnormal heartbeat stage: As the duration of pathogen stress increases, the heartbeat waveform of abalone begins to become abnormal, showing irregular waveforms and occasional cardiac arrest. The heart rate frequency decreases linearly with the increase in exposure time ( Figure 3 ), establish a linear regression equation between Bao's heart rate and time in this stage (in this example, y = -0.2092x + 189.22);

[0057] c. Calculate the horizontal axis of the intersection of the two linear equations, which is the turning point when Bao's heartbeat changes from normal to abnormal ( Figure 3 The intersection point 1 in the experiment was 633.33 min, and the time of bacteria injection (200 min in this example) was subtracted from this time. The resulting time was defined as the cardiac break timepoint upon bacteria (CBTB, 413.33 min in this example). CBTB was used to evaluate the adaptability of the experimental abalone to bacteria stress.

[0058] d. According to the linear equation of heart rate and time in the abnormal heartbeat stage, calculate the horizontal axis (time, Figure 3The intersection 2 in the figure (904.49 min in this example) is subtracted from the above CBTB, and the resulting time is defined as the cardiac zero timepoint upon bacteria (CBZB, 217.16 min in this example). CZTB represents the rate at which the heart rate of the experimental abalone decreases during the process of pathogen stress.

[0059] Combining the two abalone pathogen tolerance evaluation indicators, CBTB and CZTB, can evaluate the tolerance of abalone heartbeat function under pathogen stress. Generally speaking, the larger the CBTB, the stronger the abalone heartbeat's ability to adapt to pathogens; the larger the CZTB, the stronger the abalone's ability to resist heart rate drop under pathogen stress.

[0060] Experimental example:

[0061] In order to verify the above-mentioned heartbeat-based abalone disease resistance evaluation method, the new hybrid abalone variety "Haliotis gigantea × Haliotis discus hannai, GD, Ministry of Agriculture New Variety Certificate No. GS-02-008-2014" with typical disease resistance, and its paternal group Haliotis discus hannai (Haliotis discus hannai, DD) and maternal group Haliotis gigantea (Haliotis gigantea, GG) three abalone genotypes were used as test objects. Preliminary experiments showed that after a concentration of 2×10 9 cfu / mL of abalone pathogenic bacteria-Vibrio harveyi, the 96h mortality of Abalone occidentalis GD, Abalone DD and Abalone occidentalis GG were 53.33±11.55%, 93.33±11.55% and 93.33±5.77% respectively. The mortality of GD after pathogen stress was significantly lower than that of its parents DD and GG. In order to study and compare the differences in cardiac function of three genotypes of Abalone variegated during pathogen exposure, we injected 50uL of 2×10 9 cfu / mL of Vibrio harveyi, and recorded the real-time heartbeat of the experimental abalone throughout the process and calculated its heart rate frequency. According to this method, 24 abalone of each abalone genotype (a total of 72 abalone) were tested for the changes in heartbeat under pathogen stress, and the CBTB and CZTB indicators of the three abalone genotypes were calculated. The results are shown in the attached Figure 4As shown, the average CBTB value of the GD population (903.00±32.92min) is significantly higher than that of the parent DD population (455.00±70.31min) and GG population (399.00±56.72min), indicating that it takes a longer time to reach the heart rate inflection point under pathogen stress, and it is speculated that it has a higher immunity to pathogen stress; and the average CZTB of the GD population (1013.00±66.81min) is significantly higher than that of DD (715.00±143.05min) and GG (735.67±136.53min), indicating that the rate of decline of its heart rate after the inflection point is also relatively slow, and its resistance to pathogen invasion is higher. The high CBTB and CZTB of GD are significantly positively correlated with its lower survival rate of Vibrio infection, showing heterosis of disease resistance. The results show that the two indicators CBTB and CZTB developed based on the implementation method of the present invention can well indicate the heartbeat performance of abalone under pathogen stress, and can evaluate the immune disease resistance of the abalone to be tested in real time, accurately, intuitively and quantifiably.

[0062] Finally, all the parts not described in the present invention adopt mature products and mature technical means in the prior art.

[0063] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for evaluating abalone disease resistance based on infrared heartbeat detection, characterized in that: The following steps are involved: Step 1: The abalone to be tested is domesticated under controlled acclimation conditions indoors for 7-14 days to adapt it to the laboratory environment; Step 2: Connect the abalone heartbeat detection device before the test; Step 3: clean the attachments on the surface of the abalone shell and wipe off the moisture; Turn on the power of the Powerlab signal receiver of the abalone heartbeat detection device and the computer, set the LabChart software in the computer to the signal acquisition state, press the infrared probe of the abalone heartbeat detection device against the abalone shell surface at the abalone heart position, observe the heartbeat waveform state detected in the LabChart software at the same time, and fine-tune the position of the infrared probe to make the heartbeat waveform regular and clear; then use strong adhesive glue to stick the infrared probe to the corresponding shell surface position; Step 4: Place the abalone with the probe attached into a temporary storage tank and store it at 20-25°C for 2-4 hours to adapt it to the detection environment; Step 5: inject 50-100uL of abalone pathogenic bacteria from the upper foot of the experimental abalone; Step 6: During the pathogen stress process, the abalone heartbeat signal was recorded in real time using LabChart software until the heartbeats of all experimental abalones stopped beating normally, and the recording was terminated. After the collection was completed, the abalone heart rate was statistically analyzed; Step 7: According to the heartbeat law of abalone during the process of pathogen stress, the inflection point time when the abalone heart rate starts to decrease minus the time of pathogen injection is defined as the inflection point of the abalone pathogen stress heart rate, and the time when the abalone heart rate drops to zero is the heart rate zeroing point under pathogen stress; combining the two indicators of CBTB and CZTB, comprehensively reflecting the tolerance of the abalone heartbeat function under pathogen stress, thereby evaluating the disease resistance of abalone; the larger the CBTB value, the stronger the adaptability of the abalone heartbeat to pathogens; The larger the CZTB value is, the stronger the ability of abalone to resist the decrease in heart rate under the stress of pathogens is.

2. A method for evaluating abalone disease resistance based on infrared heartbeat detection according to claim 1, characterized in that: In the step 1, the shell length of the abalone to be tested is not less than 2.0 cm; the acclimation conditions include: temperature 20-25°C, salinity 30-33psu, pH 7.8-8.2, dissolved oxygen ≥5.0mg / L, continuous aeration of the aquaculture water, full water replacement every day, feeding fresh Gracilaria, kelp or laver during the acclimation period, and timely cleaning of residual bait, feces, pseudo-feces, and dead abalone.

3. A method for evaluating abalone disease resistance based on infrared heartbeat detection according to claim 1, characterized in that: The abalone heartbeat detection device in step 2 includes: a temporary holding tank, a heat preservation tank, a Powerlab signal receiver, an AMP03 signal amplifier, an infrared probe, a temperature-controlled circulating water bath, a temperature recorder, a computer, and LabChart software; the temporary holding tank is placed in the heat preservation tank, the seawater in the heat preservation tank is connected to the temperature-controlled circulating water bath through a water pipe, and the water temperature in the temporary holding tank is monitored and recorded by the temperature recorder to control the temperature of the seawater in the temporary holding tank; the infrared probe is fixed on the shell surface at the heart position of the abalone, and then connected to the AMP03 signal amplifier through an RJ11 connection, and the AMP03 signal amplifier is connected to the Powerlab signal receiver through a BNC method to capture, amplify and collect the abalone heartbeat signal; the Powerlab signal receiver is connected to the computer through a USB method, and the collected heartbeat signal is visualized and the heart rate is analyzed through the LabChart software.

4. A method for evaluating abalone disease resistance based on infrared heartbeat detection according to claim 1, characterized in that: In the step three, the attachments are attached organisms, dirt, mucous membranes, etc.; the strong adhesive glue is 502 glue or Blu-Tag blue tack glue.

5. A method for evaluating abalone disease resistance based on infrared heartbeat detection according to claim 1, characterized in that: In step 5, the injection concentration of the abalone-derived pathogenic bacteria is the 96-hour minimum 100% lethal concentration of the experimental abalone population.

6. A method for evaluating abalone disease resistance based on infrared heartbeat detection according to claim 1, characterized in that: In step 7, the heartbeat pattern of the abalone during the pathogen stress process is divided into two stages according to its characteristics: a. Normal heartbeat stage: the abalone's heartbeat waveform is normal and the heartbeat frequency is stable; b. Abnormal heartbeat stage: The heartbeat waveform of Bao begins to become abnormal, manifested as irregular waveform, occasional cardiac arrest, and a decreased heart rate.

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