A method for rapidly culturing trichodinids

Abstract

The application discloses a method for rapidly culturing trichodinid, and belongs to the field of aquatic biological technology. Trichodinid widely parasitizes on the body surface of many marine and freshwater economic fishes, is one of main parasitic pathogenic agents of the cultured fishes, is easily transmitted by indirect contact between hosts, causes large-scale death of fry, and brings huge economic losses to fish culture industry. However, trichodinid cannot be cultured in vitro, and its natural infection has seasonal regularity, so that the acquisition of experimental materials of trichodinid has great limitations, the research on trichodinid has been less, and the development of trichodinid disease prevention and treatment drugs and vaccines is hindered. The application achieves the effect that the trichodinid parasitizing on the body surface of the young fish with a certain initial trichodinid infection amount is massively reproduced in a short time by injecting an immunosuppressive agent into the young fish. The method is simple in operation, can significantly shorten the experimental period of trichodinid related research, and is beneficial to the development of drugs and vaccines for preventing and treating trichodinid disease.

Description

Technical Field

[0001] This invention relates to the field of aquatic organism technology, and more specifically, to a method for rapidly culturing third-generation worms. Background Technology

[0002] Gyrodactylids belong to the phylum Platyhelminthes, class Monogenea, order Gyrodactylidea, family Gyrodactylidae, and genus Gyrodactylus. They primarily parasitize the body surface and gills of fish, and are widely distributed in both seawater and freshwater environments worldwide, infecting the vast majority of fish species. The worms are roughly spindle-shaped, typically 0.3–0.8 mm in length. They are obligate parasites with host specificity, and over 400 species have been reported, with new species continuously being discovered in recent years.

[0003] The reproductive mode of Gyrodactylus is viviparous, and it has unique hyperviviparous reproduction and larval reproduction capabilities. It can reproduce rapidly in a short period of time, and Gyrodactylus can easily spread laterally through inter-host contact. While causing serious harm to farmed fish, it can also serve as a good model for studying the interaction mechanism between parasites and host fish.

[0004] The optimal temperature for the survival of Gyrodactylus is generally around 20℃. Gyrodactylus infection is prevalent in late spring and early summer, posing a significant threat to fish fry and fingerlings. It can cause skin injuries, leading to secondary infections by bacteria and other pathogens. After fish become infected, abnormal mucus secretion, congestion, and petechial hemorrhages are observed. A dull, grayish-blue mucus film forms on the skin of diseased fish. They become extremely restless, swimming frantically or rubbing their bodies against the pond walls. They also experience loss of appetite, emaciation, and eventually death. Microscopic examination often reveals the parasite on the body surface, fins, and gills of infected fish.

[0005] The salmonid parasite *Gyrodactylus salaris* has devastated wild Atlantic salmon (Salmo salar) populations in Norway and has shown a trend of expanding its spread in recent years, seriously affecting the development of farmed salmon and causing huge economic losses to fish farmers. According to reports in recent years, the parasite has caused continuous damage to various economically important fish species farmed in my country, including crucian carp (Carassius auratus), snakehead (Channa argus), tilapia (Oreochromis niloticus), carp (Cyprinus carpio), and loach (Misgurnus anguillicaudatus). Mass infections of *Gyrodactylus kobayashi* caused large-scale deaths of goldfish (Carassius auratus) in aquarium markets in Henan Province, my country.

[0006] However, since Gyrodactylus can not yet be cultured in vitro and laboratory culture is very difficult, researchers have difficulty obtaining enough samples at any time. Therefore, there is very little research on Gyrodactylus, especially on the immune interaction between Gyrodactylus and host fish, which greatly hinders the development of related drugs and vaccines.

[0007] Currently, only one experimental model of Gyrodactylus trigeminate has been reported in China: the "Gyrodactylus kobayashi-goldfish" infection experimental model (Tu Xiao, 2019. Study on the action and mechanism of arctiinogen on the target of monogenean trematodes in fish. Northwest A&F University). Its main steps are: obtaining parasite-free goldfish through praziquantel soaking, artificial inoculation of Gyrodactylus kobayashi into the tail fin, identification of Gyrodactylus species, and population maintenance. The main drawback of this model is:

[0008] (1) The abundance of Gyrodactylus infection has a regular pattern of first rising and then falling. It usually reaches the peak of infection in about 7 days and the abundance of infection drops to the lowest point or even 0 in about 14 days. Therefore, it takes at least 7 days to obtain a large amount of Gyrodactylus from a batch of goldfish. In addition, to maintain the population size of Gyrodactylus, some new uninfected goldfish must be added within 14 days, which greatly increases the time and economic costs and makes it impossible to obtain a large amount of Gyrodactylus material at any time according to demand.

[0009] (2) There are at least 7 species of Gyrodactylus that can parasitize goldfish, so it is difficult to maintain a single species of Gyrodactylus. In the case of ensuring that the Gyrodactylus obtained from cultivation is a single species, each time goldfish are added to maintain the population, the process of killing the parasite and restoring the population must be carried out first. If the preservation is interrupted by accident, the preservation process of single species identification and single species cultivation of Gyrodactylus must be carried out again, which increases the difficulty of operation and time cost. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for rapidly culturing third-generation worms.

[0011] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0012] A method for rapidly culturing third-generation worms includes the following steps:

[0013] S1. Select juvenile fish with an initial infection level of 5-10 Gyrodactylus to obtain the selected juvenile fish;

[0014] S2. Inject the selected juvenile fish as described in step S1 with an immunosuppressant to obtain the injected juvenile fish;

[0015] S3. Culture the injected juvenile fish until the amount of Gyrodactylus infection in the injected juvenile fish is equal to or exceeds the target multiple of the Gyrodactylus infection amount described in step S1, and the culture process ends; the culture is carried out in aquaculture water that does not contain pathogenic microorganisms in the juvenile fish, and the water temperature is 24-26℃.

[0016] Preferably, the third-generation worm is *Gyrodactylus cristatus*.

[0017] Preferably, the juvenile fish mentioned in step S1 is a tilapia juvenile.

[0018] More preferably, the juvenile fish mentioned in step S1 is a juvenile tilapia, a juvenile Galilean tilapia, a juvenile Nile tilapia, or a juvenile Mozambique tilapia.

[0019] Most preferably, the juvenile fish mentioned in step S1 is a juvenile Nile tilapia.

[0020] Based on the inventor's preliminary research, only one species of Gyrodactylus parasite, Gyrodactylus kobayeri, was identified as a host fish in Nile tilapia juveniles. Given the need to ensure that the cultured Gyrodactylus is a single species, using Nile tilapia juveniles as the host fish in the Gyrodactylus culture process avoids the complex operations of frequent Gyrodactylus identification and single-species preservation required in the "goldfish-Kobayashi Gyrodactylus" model, greatly improving experimental efficiency. Furthermore, the required host fish are common and inexpensive.

[0021] Preferably, the specifications of the juvenile fish in step S1 are: body length 3-5cm and weight 1-3g.

[0022] Preferably, the method for preparing juvenile fish with an initial infection dose of 5-10 Gyrodactylus is as follows: healthy juvenile fish are placed in a disinfected rearing tank, and fins carrying Gyrodactylus are introduced into the rearing tank. After 12-36 hours, the fins are removed, and the infection status of Gyrodactylus in the healthy juvenile fish is examined under a microscope every 12-36 hours. Juvenile fish with an infection dose of 5-10 Gyrodactylus are selected.

[0023] Preferably, in step S2, the immunosuppressant is dexamethasone, a pharmaceutically acceptable salt of dexamethasone, tacrolimus, a pharmaceutically acceptable salt of tacrolimus, rapamycin, or a pharmaceutically acceptable salt of rapamycin.

[0024] More preferably, in step S2, the immunosuppressant is dexamethasone or a pharmaceutically acceptable salt of dexamethasone.

[0025] Most preferably, in step S2, the injection dosage is 0.1 mg of dexamethasone sodium phosphate per gram of fish body weight.

[0026] More preferably, in step S2, the immunosuppressant is dexamethasone sodium phosphate at a concentration of 1-5 mg / mL.

[0027] As a reference for specific implementation, the juvenile fish selected in step S1 were injected with a concentration of 5 mg / mL of veterinary dexamethasone sodium phosphate injection at an injection volume of 20 μL per gram of fish body weight.

[0028] Preferably, in step S2, the injection site is the base of the pelvic fin.

[0029] As a reference for specific implementation, in step S2, after anesthetizing the juvenile fish selected in step S1, anesthetize the fish by inserting a needle into the abdominal cavity at an angle of about 45 degrees at the base of the pelvic fin to inject an immunosuppressant. After the injection, leave the needle in place for more than 5 seconds before pulling it out.

[0030] Preferably, in step S3, the surface of the juvenile fish is observed every 12 to 36 hours until the amount of Gyrodactylus infection in the juvenile fish after injection is equal to or exceeds the target multiple of the Gyrodactylus infection amount described in step S1, at which point the culture process ends.

[0031] More preferably, in step S3, the target multiple is not higher than the highest infection level of Gyrodactylus in the juvenile fish.

[0032] Most preferably, in step S3, the target multiple is 10 times.

[0033] Preferably, in step S3, the aquaculture water that does not contain pathogenic microorganisms of the juvenile fish is aquaculture water that has been sterilized and aerated.

[0034] More preferably, in step S3, the cultivation involves raising the injected juvenile fish separately in sterilized and aerated aquaculture water.

[0035] The application of the method in constructing the "Gyrodactylus-Tilapia" infection model should also be within the scope of protection of this invention.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) This invention enables rapid and large-scale reproduction of the third generation of insects in the laboratory. Usually, the number of third generation insects can be increased by 10 times or more within 3 days, which makes it more convenient and faster to apply to experiments. The method of claiming protection by this invention can obtain a large number of third generation insects for experiments as needed, which greatly shortens the experimental cycle of third generation insect-related research and solves the problems of no insects available and long experimental cycles in third generation insect-related research.

[0038] (2) The operation method claimed in this invention is simple and easy to implement. During the cultivation of the third generation of parasites, each key link can be controlled and adjusted to better ensure the quantity, quality and stability of the cultivated third generation of parasites. This solves the problem that researchers have difficulty obtaining third generation of parasite resources stably, which is conducive to the development of drugs and vaccines for the prevention and treatment of third generation of parasites. Attached Figure Description

[0039] Figure 1 This describes the temporary rearing process of Nile tilapia after injection.

[0040] Figure 2 A stereomicroscopic image (25×) of *Gyrodactylus cristatus* that proliferates in large numbers on the caudal fin of Nile tilapia. The red arrows and all translucent rod-shaped objects in the image are *Gyrodactylus cristatus*.

[0041] Figure 3 The infection rate and intensity of *Gyrodactylus gracilis* in Nile tilapia juveniles were determined at different temporary holding temperatures. * indicates a significant difference from other groups (P<0.05). Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0043] Example 1: A method for rapidly culturing third-generation worms

[0044] I. Experimental Methods

[0045] A method for rapidly culturing third-generation worms, the main steps of which include:

[0046] S1. Select juvenile Nile tilapia with a body length of 3-5cm and a weight of about 1-3g from the purchased Nile tilapia. Examine the juvenile Nile tilapia under a stereomicroscope and select juvenile Nile tilapia with an infection level of 5-10 Gyrodactylus larvae to obtain 20 juvenile Nile tilapia with a small amount of Gyrodactylus larvae.

[0047] S2. At an injection dose of 20 μL per gram of fish, the Nile tilapia juveniles that were slightly infected with Gyrodactylus trichodiniae obtained in step S1 were injected with immunosuppressants. The immunosuppressant used was veterinary dexamethasone sodium phosphate injection solution with a concentration of 5 mg / mL. After anesthetizing the Nile tilapia juveniles that were slightly infected with Gyrodactylus trichodiniae, the needle was inserted into the abdominal cavity at an angle of about 45 degrees at the base of the pelvic fin for intraperitoneal injection. The needle was left in place for more than 5 seconds after injection before being withdrawn to prevent leakage. The injected Nile tilapia juveniles were obtained.

[0048] S3. For example Figure 1 As shown, the Nile tilapia fry obtained after injection in step S2 are placed separately in plastic boxes for temporary rearing. The water temperature is strictly controlled at 24-26℃. The water used for temporary rearing must be sterilized and aerated aquaculture water to prevent the introduction of pathogens such as bacteria.

[0049] S4. After injection, examine the surface of the Nile tilapia fry using a stereomicroscope every 24 hours. The culture process ends when the amount of Gyrodactylus infection on the surface of the Nile tilapia fry is equal to or exceeds 10 times the amount of Gyrodactylus infection described in step S1.

[0050] II. Experimental Results

[0051] In 90% of Nile tilapia juveniles, the number of Gyrodactylus cells on their body surface increased to more than 10 times the original Gyrodactylus infection level within 3 days. Figure 2 As shown, the cichlid parasites on the body surface of juvenile Nile tilapia are mainly concentrated on the caudal fin. Figure 2 The red arrows and all the translucent rod-shaped objects are Cichlididae 3rd generation.

[0052] The Nile tilapia infected with Gyrodactylus cristatus obtained by this method can be used for subsequent experiments, including Gyrodactylus biological research, Gyrodactylus infection experiments, Gyrodactylus drug effects research, Gyrodactylus protein function research, and immune interaction research between Gyrodactylus cristatus and host fish, etc.

[0053] Example 2: A method for rapidly culturing third-generation worms

[0054] I. Experimental Methods

[0055] A method for rapidly culturing Gyrodactylus is provided. The main steps are the same as those in Example 1, Section I, Experimental Methods. The difference is that in step S1, juvenile Nile tilapia with a body length of 3-5 cm and a weight of about 1-3 g are selected. The juvenile Nile tilapia are examined under a stereomicroscope. Healthy juvenile Nile tilapia that are not infected with Gyrodactylus ciliata are selected. After infecting them with Gyrodactylus ciliata preserved in the laboratory, juvenile Nile tilapia with an infection level of 5-10 Gyrodactylus ciliata are selected to obtain 20 juvenile Nile tilapia that have been slightly infected with Gyrodactylus ciliata.

[0056] The detailed steps for infecting Nile tilapia with Gyrodactylus are as follows: Before the experiment, disinfect the circulating water system, select healthy Nile tilapia juveniles that are not infected with Gyrodactylus, and raise them in the disinfected pool. Put the fins of fish infected with Gyrodactylus into the pool, stop water circulation, and remove the fins from the pool after 24 hours. Observe the Gyrodactylus infection level of the Nile tilapia juveniles every 24 hours. Select Nile tilapia juveniles with 5 to 10 Gyrodactylus infections to obtain Nile tilapia juveniles with a small amount of Gyrodactylus infection.

[0057] II. Experimental Results

[0058] The number of Gyrodactylus larvae on the body surface of 80% of Nile tilapia juveniles increased to more than 10 times the original Gyrodactylus infection level within 3 days.

[0059] Comparative Example 1: Effect of different initial infection doses of Gyrodactylus on the culture effect of Gyrodactylus.

[0060] I. Experimental Methods

[0061] The experimental method is the same as in "I. Experimental Method" of Example 1, except that:

[0062] In step S1, juvenile Nile tilapia were examined under a stereomicroscope, and experimental groups with different initial infection levels of Gyrodactylus were set up:

[0063] Five juvenile Nile tilapia with an initial infection level of less than 5 Gyrodactylus were selected and designated as experimental group 1.

[0064] Five juvenile Nile tilapia with an initial infection level of more than 10 Gyrodactylus were selected and designated as experimental group 2.

[0065] Nile tilapia juveniles with different initial infection levels of Gyrodactylus;

[0066] In step S4, the mortality rate of Nile tilapia was counted within three days of injection, and the surface of the Nile tilapia fry was examined every 24 hours using a stereomicroscope.

[0067] II. Experimental Results

[0068] Table 1 shows the infection status of Gyrodactylus in Nile tilapia in different experimental groups within three days after injection. As shown in Table 1, in Nile tilapia juveniles with an initial Gyrodactylus infection of less than 5 individuals (experimental group 1), 40% of the host fish cichlids showed a Gyrodactylus infection increase of more than 10 times within three days after injection. However, due to the low initial Gyrodactylus infection level in the fish, the host fish's Gyrodactylus infection level was still insufficient to meet the requirements of subsequent experiments. In Nile tilapia juveniles with an initial Gyrodactylus infection of more than 10 individuals (experimental group 2), the mortality rate was 20% after three days of injection. This indicates that an excessively high initial Gyrodactylus infection level can easily lead to the death of the host fish after injection of immunosuppressants. Furthermore, in experimental group 2, the growth rate of Gyrodactylus in the host fish was slower after injection of immunosuppressants, with only 20% of the host fish cichlids showing a Gyrodactylus infection increase of more than 10 times within three days. Therefore, the optimal initial Gyrodactylus infection level was determined to be 5–10 individuals.

[0069] Table 1. Statistics on Gyrodactylus infection status in Nile tilapia from different experimental groups within three days after injection.

[0070]

[0071] Example 3: Effect of different fish sizes on the culture effect of the third generation of parasites

[0072] I. Experimental Methods

[0073] The experimental method is the same as in "I. Experimental Method" of Example 1, except that:

[0074] In step S1, the juvenile Nile tilapia were examined under a stereomicroscope. Juvenile Nile tilapia with 5–10 Gyrodactylus infections were selected, and two experimental groups of Nile tilapia of different body lengths were established:

[0075] Eight juvenile Nile tilapia with a body length of less than 3 cm and a weight of about 0.2 to 1 g were selected and designated as experimental group 1.

[0076] Eight juvenile Nile tilapia, measuring 3–5 cm in length and weighing approximately 1–3 g, were selected and designated as experimental group 2.

[0077] Nile tilapia fry with a small amount of infection with Gyrodactylus cristatus were obtained;

[0078] In step S4, the mortality rate of Nile tilapia was counted three days after injection, and the surviving juvenile Nile tilapia were examined under a stereomicroscope.

[0079] II. Experimental Results

[0080] Because juvenile Nile tilapia with a body length greater than 5cm have a more complete immune system, they are less likely to be infected by Gyrodactylus cristatus. Therefore, it is not possible to select a sufficient number of host fish for experiments in the initial screening. Moreover, the individuals are too large to be suitable for microscopic examination. Thus, juvenile Nile tilapia with a body length greater than 5cm are not selected for experiments.

[0081] Table 2 shows the infection status of Gyrodactylus in Nile tilapia juveniles in different experimental groups three days after injection of 5 mg / mL veterinary dexamethasone sodium phosphate injection. As shown in Table 2, the mortality rate of Nile tilapia juveniles with a body length of less than 3 cm (experimental group 1) was as high as 50% three days after injection, and the amount of Gyrodactylus in the fish was relatively small; the mortality rate of Nile tilapia juveniles with a body length between 3 and 5 cm (experimental group 2) was 0% within three days. Within three days after injection, 75% of the juveniles had a Gyrodactylus infection level more than 10 times the original infection level. After three days, more than 50% of the fish still maintained a Gyrodactylus number more than 10 times the original infection level. Since the abundance of Gyrodactylus infection has a regular pattern of first rising and then falling, the infection peak of Nile tilapia juveniles with a body length of 3-5 cm can be reached within three days, and then it will drop. The data in Table 2 show that Nile tilapia juveniles with a body length of 3-5 cm can still provide a large amount of Gyrodactylus for subsequent experiments three days after injection.

[0082] Table 2. Statistics on Gyrodactylus infection status in Nile tilapia from different experimental groups three days after injection.

[0083]

[0084] Example 4: Effect of different immunosuppressant injection doses on the culture effect of Gyrodactylus triglavius.

[0085] I. Experimental Methods

[0086] The experimental method is the same as in "I. Experimental Method" of Example 1, except that:

[0087] In step S2, experimental groups with different injection volumes are set up:

[0088] Experimental group 1: Ten juvenile Nile tilapia that had been slightly infected with Gyrodactylus cristatus in step S1 were selected and injected with immunosuppressants at a dose of 10 μL per gram of fish.

[0089] Experimental group 2: Ten juvenile Nile tilapia that had been slightly infected with Gyrodactylus cristatus in step S1 were selected and injected with immunosuppressants at a dose of 20 μL per gram of fish.

[0090] Experimental Group 3: Nine juvenile Nile tilapia with a small amount of *Gyrodactylus* infection obtained in step S1 were injected with immunosuppressants at a dose of 30 μL per gram of fish.

[0091] The immunosuppressant used was veterinary dexamethasone sodium phosphate injection with a concentration of 5 mg / mL. After anesthetizing the Nile tilapia juveniles that had been slightly infected with Gyrodactylus, the needle was inserted into the abdominal cavity at an angle of about 45 degrees at the base of the pelvic fin for intraperitoneal injection. The needle was left in place for more than 5 seconds after injection before being withdrawn to prevent leakage. The injected Nile tilapia juveniles were obtained.

[0092] In step S4, the mortality rate of Nile tilapia was counted within three days of injection, and the surface of the Nile tilapia fry was examined every 24 hours using a stereomicroscope.

[0093] II. Experimental Results

[0094] Table 3 shows the infection status of Gyrodactylus in Nile tilapia juveniles in different experimental groups within three days after injection. Since the highest infection rate may not occur on the third day after injection, it may also reach its peak on the first or second day after injection. Therefore, the highest growth multiple of infection rate within three days after injection is statistically analyzed.

[0095] As shown in Table 3, in experimental group 1 where the injection dose was 10 μL per gram of fish, only one juvenile Nile tilapia showed a maximum increase in infection rate of more than 10 times within three days after injection.

[0096] In experimental group 2, where the injection dose was 20 μL per gram of fish, 60% of the Nile tilapia juveniles showed a maximum increase in infection rate of more than 10 times within three days after injection.

[0097] In experimental group 3, where the injection dose was 30 μL per gram of fish, more than 50% of the Nile tilapia fry died within three days after injection before they had developed a large number of Gyrodactylus cells.

[0098] Therefore, the optimal injection dose was determined to be 20 μL of veterinary dexamethasone sodium phosphate injection at a concentration of 5 mg / mL per gram of fish.

[0099] Table 3. Statistics on Gyrodactylus infection status in Nile tilapia juveniles within three days after injection in different injection dose groups.

[0100]

[0101]

[0102] Example 5: Effect of different injection sites on the culture effect of Gyrodactylus larvae

[0103] I. Experimental Methods

[0104] The experimental method is the same as in "I. Experimental Method" of Example 1, except that:

[0105] In step S2, 20 μL of immunosuppressant was injected into the Nile tilapia juveniles obtained in step S1 that had been slightly infected with Gyrodactylus tricuspidata. The immunosuppressant used was veterinary dexamethasone sodium phosphate injection at a concentration of 5 mg / mL. The Nile tilapia juveniles that had been slightly infected with Gyrodactylus tricuspidata were anesthetized before injection. Experimental groups with different immunosuppressant injection sites were set up:

[0106] Experimental group 1: Five Nile tilapia juveniles that had been infected with a small amount of Gyrodactylus in step S1 were selected and injected into the mid-peritoneal cavity.

[0107] Experimental group 2: Five Nile tilapia juveniles that had been infected with a small amount of Gyrodactylus in step S1 were selected and injected into the dorsal muscle.

[0108] Experimental group 3: Five juvenile Nile tilapia with a small amount of infection with Gyrodactylus cristatus obtained in step S1 were selected and injected intraperitoneally at the base of the pelvic fin.

[0109] After injection, wait at least 5 seconds before pulling out the needle to prevent leakage and obtain Nile tilapia fry after injection;

[0110] In step S4, the mortality rate of Nile tilapia was counted within three days of injection, and the surface of the Nile tilapia fry was examined every 24 hours using a stereomicroscope.

[0111] II. Experimental Results

[0112] Table 4 shows the infection status of Gyrodactylus in cichlids within three days after injection at different injection sites. As shown in Table 4, injection of immunosuppressants in the middle of the abdominal cavity (experimental group 1) easily led to the death of the host fish; injection of immunosuppressants into the dorsal muscle (experimental group 2) had poor effect, with only one juvenile Nile tilapia showing an increase in Gyrodactylus infection exceeding 10 times within three days; while intraperitoneal injection at the base of the pelvic fin (experimental group 3) not only effectively prevented the death of the host fish, but also resulted in a Gyrodactylus infection exceeding 10 times within three days. Therefore, the optimal injection site was determined to be intraperitoneal injection at the base of the pelvic fin.

[0113] Table 4. Statistics on Gyrodactylus infection in cichlids within three days after injection at different injection sites.

[0114]

[0115]

[0116] Example 6: Effect of different temporary rearing water temperatures on the culture effect of the third generation of worms

[0117] I. Experimental Methods

[0118] The experimental method is the same as in "I. Experimental Method" of Example 1, except that:

[0119] In step S3, experimental groups with different temporary holding water temperatures were set up:

[0120] Experimental group 1: 30 Nile tilapia fry obtained in step S2 after injection were randomly selected and placed in plastic boxes for temporary rearing, with the water temperature strictly controlled at 18℃;

[0121] Experimental group 2: 30 Nile tilapia fry obtained in step S2 after injection were randomly selected and placed in plastic boxes for temporary rearing, with the water temperature strictly controlled at 25℃;

[0122] Experimental Group 3: Thirty Nile tilapia fry obtained in step S2 after injection were randomly selected and temporarily housed in plastic boxes, with the water temperature strictly controlled at 32℃.

[0123] The water used for temporary holding must be sterilized and aerated aquaculture water to prevent the introduction of pathogens such as bacteria.

[0124] II. Experimental Results

[0125] Figure 3 To determine the infection rate and intensity of *Gyrodactylus gracilis* infection in juvenile Nile tilapia at different temporary holding temperatures, the following methods were used: Figure 3 It can be seen that the infection rate and infection intensity of experimental group 2 (water temperature of 25℃) were significantly higher than those of experimental group 1 (water temperature of 18℃) and experimental group 3 (water temperature of 32℃), and the efficiency of obtaining the third generation of parasites was the highest. Therefore, 25℃ was determined to be the optimal water temperature for the culture process of third generation parasites in cichlids.

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for rapidly culturing third-generation worms, characterized in that, Includes the following steps: S1. Select juvenile fish with an initial infection level of 5-10 Gyrodactylus to obtain the selected juvenile fish; The term "third generation worm" refers to *Cyprinus trigenidae*, and the term "juvenile fish" refers to tilapia juveniles. The specifications of the juvenile fish are: body length 3-5cm and weight 1-3g. S2. Inject the selected juvenile fish as described in step S1 with an immunosuppressant to obtain the injected juvenile fish; The immunosuppressant is dexamethasone or a pharmaceutically acceptable salt of dexamethasone, and the injection site is the base of the pelvic fin. S3. Cultivate the injected juvenile fish until the amount of Gyrodactylus infection in the injected juvenile fish is equal to or exceeds the target multiple of the Gyrodactylus infection amount described in step S1, and the cultivation process ends; the cultivation is carried out in aquaculture water that does not contain pathogenic microorganisms in the juvenile fish, and the water temperature is 24-26℃; the cultivation is carried out by raising each injected juvenile fish individually in sterilized and aerated aquaculture water.

2. The method according to claim 1, characterized in that, In step S1, the method for preparing juvenile fish with an initial infection level of 5 to 10 Gyrodactylus is as follows: healthy juvenile fish are placed in a disinfected rearing tank, and fins carrying Gyrodactylus are introduced into the rearing tank. After 12 to 36 hours, the fins are removed, and the infection status of Gyrodactylus is examined under a microscope every 12 to 36 hours. Juvenile fish with an infection level of 5 to 10 Gyrodactylus are selected.

3. The method according to claim 1, characterized in that, In step S2, the injection dosage is 0.1 mg of dexamethasone sodium phosphate per gram of fish body weight.

4. The method according to claim 1, characterized in that, In step S3, the juvenile fish are observed every 12 to 36 hours after injection. The culture process ends when the amount of Gyrodactylus infection in the juvenile fish is equal to or exceeds the target multiple of the amount of Gyrodactylus infection described in step S1.

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