A method for sampling live abalone

Abstract

The application discloses a kind of live body sampling method of east wind snail, comprising: selecting healthy and normal vigor east wind snail, place in the container that is sterilized using potassium permanganate in advance, using 2.5% seawater alcohol anesthesia, in anesthetized state, with sterilized scissors take half of water pipe or the rear of foot muscle, any part of side, about equal to half of water pipe size, then using 75% alcohol disinfection, then apply erythromycin ointment, place in ultraviolet sterilized seawater and relieve anesthesia, then sand culture for 7 days, sand is laid on the seventh day and is fed, if snail can normally drill sand and eat, then it indicates that live body sampling is basically successful. About 3 weeks, new tissue can grow and grow into about 5 weeks, wound is completely recovered after growing, and east wind snail survival state can be completely recovered normal. The method can make the survival rate of east wind snail live body sampling reach more than 95%, and the sample can be used for resequencing for the snail with a body length of more than 2 cm, which is important for the development of various experiments such as east wind snail breeding.

Description

Technical Field

[0001] This invention relates to the field of shellfish sampling technology, specifically to a method for sampling live snails. Background Technology

[0002] The African whelk (Babylonia areolata) belongs to the phylum Mollusca, class Gastropada, subclass Prosobranchia, order Nerobanchia, and family Babyloniidae. Commonly known as the flower whelk, yellow whelk, or phoenix whelk, it is an aquatic animal with extremely high nutritional value. The aquaculture industry of the spotted African whelk (Babylonia areolata) has reached a certain scale in Hainan, Fujian, and Guangdong provinces. Selective breeding work on its growth traits, disease resistance, food conversion rate, and low-salt tolerance, which are important economic traits, is underway and has made some progress. However, for a long time, the low survival rate after sampling has been a problem in African whelk experiments. That is, after resequencing and other experiments, the individual may not survive, making it difficult to pass on its superior traits to the next generation. This has adversely affected African whelk breeding work and the conduct of various related experiments.

[0003] If the individuals cannot survive or have a low survival rate after sampling, the cost of resequencing the sampled individuals during the genomic selection breeding process is high. Individuals with superior traits cannot be passed on to other generations and can only be passed on to other individuals from the same family. Such a breeding process will result in low genetic gain and high cost.

[0004] Based on this, the purpose of this invention is to provide a live sampling method with a high survival rate for the snail *Bambusa serpentina*, and to make the sampling size meet the requirements of experiments such as resequencing, which plays a particularly important role in the development of scientific research such as breeding. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a live sampling method for *Bellamya spp.* with a high survival rate.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for sampling live *Bellamya aegyptiaca* includes:

[0008] Step 1): Select healthy and vigorous snails, place them in a pre-sterilized container, and anesthetize them with 2.5% seawater alcohol.

[0009] Step 2): After the foot muscles are stretched and the animal is under anesthesia, use sterilized tools (such as scissors or blades) to take a sample from the water tube or any part of the back or side of the foot muscles. Then disinfect with alcohol, apply erythromycin ointment, and place the animal in UV-sterilized seawater to relieve anesthesia. Tissue samples taken from live *Bellamya aegypti* snails can be used in resequencing and other related experiments.

[0010] Step 3): Cultivate in sand-free seawater disinfected with ultraviolet light for 7 days, changing the water and cleaning the culture container once a day. The seawater temperature and salinity should be the same as when the anesthesia is lifted.

[0011] Step 4): On the seventh day, lay sand and feed the snails. If the snails can burrow and feed normally, it indicates that the live sampling was basically successful and they can be transferred to ordinary seawater culture. Regenerated tissue can grow in about 3 weeks and is fully grown in about 5 weeks. After it is fully grown, the wound will be completely healed and the snails can fully recover to normal living conditions.

[0012] As one possible implementation, further, the snail body selected in step 1) is more than 2cm in length.

[0013] As one possible implementation, further, the seawater used in the seawater alcohol in step 1) is disinfected with ultraviolet light in advance.

[0014] As one possible implementation, further, step 2) uses 75% alcohol for disinfection.

[0015] As one possible implementation, further, in step 2), anesthesia is relieved in ultraviolet-sterilized seawater at a temperature of 25±1℃ and a salinity of 28±1ppt.

[0016] As one possible implementation, in step 2), half of the water pipe or any part of the posterior or lateral aspect of the foot muscle is taken; wherein the sample size of the posterior or lateral aspect of the foot muscle is approximately equal to half the size of the water pipe.

[0017] As one possible implementation, the sand laid in step 4) further needs to be disinfected.

[0018] As one possible implementation, further, if the snails are not in good condition in step 4), continue to culture them in sand-free seawater after UV disinfection for 2-3 days, changing the water and cleaning the culture container once a day, with the seawater temperature and salinity being the same as when the anesthesia is lifted.

[0019] The beneficial effects of this invention are as follows:

[0020] The method of this invention can perform live sampling of *Bellamya aegyptiacus* with a survival rate of over 95%, and can satisfy repeated sampling. It can effectively overcome the problem of extremely low survival rate of existing *Bellamya aegyptiacus* sampling, and is of great help to the development of scientific research such as breeding. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the sampling site for live *Sinocyclocheilus edulis*.

[0023] Figure 2 A diagram showing the condition of a live *Sinocyclocheilus* snail after sampling.

[0024] Figure 3 This is a diagram illustrating the growth of regenerated tissue in the snail *Bellamya spp.*

[0025] Figure 4 This is a DNA quality testing diagram (Novogene Biotechnology Co., Ltd.); it indicates that the samples obtained by this sampling method can be used for resequencing. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This embodiment provides a method for sampling live *Bellamya aegyptiaca*, including the following steps:

[0028] Step 1: Randomly select healthy snails that are in normal condition, can feed normally and climb walls, and are more than 2cm in length.

[0029] Step 2: Disinfection

[0030] Take a container of suitable size that will not chemically react with potassium permanganate and seawater, add 1 ppm of potassium permanganate for disinfection, and leave it for 5 hours. If the concentration of potassium permanganate is increased, the disinfection time can be shortened accordingly. Then rinse the potassium permanganate thoroughly with fresh water.

[0031] Step 3: Anesthesia

[0032] Place the snails to be sampled in 2.5% seawater (seawater disinfected with ultraviolet light beforehand) and alcohol. They may move violently at first, so be careful to prevent them from crawling out of the container. After about 5-10 minutes, the snails will be upside down, secrete a lot of mucus, stretch their foot muscles, and move sluggishly, which indicates that the anesthesia was successful.

[0033] Step 4: Sampling

[0034] Use sterilized scissors to cut half of the snail's water pipe, or the posterior end and side of the foot muscle of a piece equal in size to half the water pipe (as shown in the attached document). Figure 1-2 As shown in the figure, after taking the sample as soon as possible, the sample was quickly placed at -80℃. The wound of the snail was disinfected with 75% alcohol and erythromycin ointment. Then, it was placed in seawater (temperature 25±1℃, salinity 28±1ppt) after UV disinfection to relieve anesthesia. The snail can recover to normal in about 5-10 minutes.

[0035] Step 5: Recovery

[0036] After the snails recover, place them in UV-sterilized seawater for further cultivation and recovery. The seawater temperature should be 25±1℃, and the salinity 28±1 ppt. For the first 7 days, do not add sand or feed them. Change the water and clean the breeding container daily. The snails may exhibit abnormal behaviors such as not being able to properly suck in their wounds while crawling, or standing upside down; these are normal. If any snails die, remove them promptly to prevent decay and the growth of pathogens that could lead to further mortality. On the seventh day, add sand and feed them. The sand must be sterilized using the same method as in step two. If the snails can burrow, feed, and climb normally after adding sand, they have recovered well and can be cultured in regular seawater. If their condition is poor, continue culturing in UV-sterilized seawater without sand for 2-3 days, changing the water daily. The seawater temperature and salinity should be the same as when they were released from anesthesia. Regenerated tissue can be observed around the 3rd week, and the wounds should be mostly healed around the 5th week, at which point the snails are fully recovered.

[0037] Once the snails have settled in the sand and are able to feed, burrow, and climb the sand walls normally, the collected samples can be sent to the company for sequencing.

[0038] Effect test

[0039] 1) The sampling method of the present invention was used to sample different parts of the whelk (the only difference between the groups was the sampling location of the live whelk; the other experimental operations and conditions were the same as those in the above embodiments), and the survival rate of the whelk after sampling was statistically analyzed. The statistical results are shown in Table 1 below:

[0040] Table 1. Survival rate of *Bellamya alatus* at different sampling sites

[0041]

[0042] As shown in the table above, the survival rate of samples taken from the foot muscle and siphon is over 95%, while the mantle sample fails to survive, and the survival rate of samples taken from the antennae is low, which also negatively impacts their feeding. Therefore, the sampling sites for the method of this invention are the posterior end of the foot muscle, the anterior and posterior sides of the foot muscle, and the siphon.

[0043] 2) The effects of different seawater treatment methods on the survival rate of *Bambusa multiplex* were tested. Specifically, the "ultraviolet-disinfected seawater" (hereinafter referred to as "ultraviolet seawater") used in steps three, four, and five of the embodiments was replaced with "sterilized seawater" and "ordinary seawater," respectively. The survival rate of *Bambusa multiplex* was then statistically analyzed, and the results are shown in Table 2 below. (Note: In the experiments described in Table 2 below, only the seawater treatment methods differed; all other experimental operations and conditions were consistent with the methods in the above embodiments.)

[0044] Table 2. Survival rate of *Bellamya aegyptiaca* under different seawater treatment methods

[0045]

[0046] Ordinary seawater is seawater that has not undergone any treatment; sterilized seawater is seawater that has been sterilized using a high-pressure steam sterilizer.

[0047] According to the data in the table above, ordinary seawater results in a high mortality rate. While seawater sterilized using a high-pressure steam sterilizer can achieve a 100% survival rate, it is also more expensive. Therefore, the seawater treatment method in this invention uses ultraviolet irradiation disinfection.

[0048] 3) The effect of different wound disinfection methods on the survival rate of *Bellamya aegyptiaca* was tested. The test results are shown in Table 3 below: (Only the wound disinfection methods differed in the experiments described in Table 3; all other experimental operations and conditions were the same as those in the above embodiments.)

[0049] Table 3. Survival rate of *Bellamya aegyptiaca* under different wound disinfection methods

[0050]

[0051] According to the data in the table above, the method of this invention can achieve a survival rate of over 95% for the snails, while simplified disinfection methods will result in a lower survival rate.

[0052] 4) The survival rate of the method of the present invention is compared with that of the traditional direct sampling method, as shown in Table 4 below:

[0053]

[0054] The traditional method involves taking samples of seawater and placing the snails directly back into the sand for rearing without treating the seawater or the wounds. This method increases the probability of wound infection, and the burrowing behavior of the snails in the sand can cause abrasion of the wounds, hindering healing.

[0055] As shown in the table above, the survival rate of snails obtained by the method of this invention is as high as 95% or more, which is much higher than that of traditional methods.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for sampling live *Bellamya aegyptiaca*, characterized in that, include: Step 1): Select healthy and vigorous snails with a body length of more than 2cm, place them in a pre-sterilized container, and anesthetize them with 2.5% seawater alcohol; the seawater used in the seawater alcohol is disinfected with ultraviolet light in advance. Step 2): After the foot muscles are stretched and the person is under anesthesia, use sterilized tools to take a sample from the back or side of the foot muscles, with the sample size from the back or side of the foot muscles being about half the size of the water pipe. Then disinfect with alcohol, apply erythromycin ointment, and place the person in seawater that has been sterilized with ultraviolet light at a temperature of 25±1℃ and a salinity of 28±1ppt to relieve anesthesia. Step 3): Cultivate in sand-free seawater disinfected with ultraviolet light for 7 days, changing the water and cleaning the culture container once a day. The seawater temperature and salinity should be the same as when the anesthesia is lifted. Step 4): On the seventh day, lay sand and feed the snails. If the snails can burrow and feed normally, it indicates that the live sampling was basically successful and they can be transferred to ordinary seawater culture.

2. The method for sampling live *Bellamya aegyptiaca* according to claim 1, characterized in that, Step 2) Use 75% alcohol for disinfection.

3. The method for sampling live *Bellamya aegyptiaca* according to claim 1, characterized in that, The sand laid in step 4) needs to be disinfected.

4. The method for sampling live *Bellamya aegyptiaca* according to claim 1, characterized in that, If the snails are not in good condition in step 4), continue to culture them in sand-free seawater that has been disinfected with ultraviolet light for 2-3 days. During this period, change the water and clean the culture container once a day. The seawater temperature and salinity should be the same as when the anesthesia is lifted.

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