A preparation method for efficiently obtaining solar long-spined sea star larvae and juvenile / larvae and application thereof

Abstract

The application discloses a preparation method and application of efficiently obtaining solar long-spined sea star larvae and juveniles / larvae. The method comprises the following steps: distinguishing the development period and the male and female genders of gonads; injecting oxytocin, separately collecting eggs and sperms, adding active sperms in batches after the eggs are broken, and the quantity ratio of the sperms and the eggs is 50-100:1; controlling the temperature, salinity and pH during the hatching of the larvae, culturing under full or half light shielding, and stirring when the fertilized eggs gather; feeding the hatched larvae with small new moon-shaped algae, changing water and removing dead and abnormal individuals; increasing the amount of feed when the larvae develop into brachiolaria, adding an attachment base when 70% of the planktonic larvae develop into late-stage brachiolaria with a size of 1000mu m*600mu m, transferring the juveniles to living stones with benthic coral algae after metamorphosis, and culturing until the long-spined sea star larvae are harvested. The application significantly improves the survival rate and metamorphosis rate of the planktonic larvae, and efficiently obtains a batch of larvae and juveniles / larvae in a short time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of marine coral reef biological breeding, and particularly relates to a preparation method for efficiently obtaining solar acanthaster larva and juvenile / larva and application thereof. BACKGROUND

[0002] In a healthy coral reef ecosystem, the long-spined sea star has the function of maintaining the structure of the coral community, but once the number is overloaded, it will cause a large number of corals to be overturned in a short time. In recent years, the reports of the outbreak of long-spined sea star have been reported one after another, and the scope and damage are increasingly obvious, which has attracted widespread attention from scholars and the public.

[0003] Although there have been a large number of studies abroad, a wealth of theoretical hypotheses have been formed, but the current understanding of the mechanism and outbreak process of the wild long-spined sea star disaster is still very insufficient. Where do a large number of long-spined sea stars come from in a short time? How does it break out? What is the outbreak mechanism? How to monitor and control? To answer these questions, the long-spined sea star larva and juvenile body are taken as the starting point, and the artificial breeding technology provides an opportunity to cultivate a batch of larvae and juvenile / larva under laboratory conditions, to simulate the growth and survival status of the larvae and juvenile bodies under the change of external environmental conditions, so as to reveal the outbreak process and mechanism of the long-spined sea star. Therefore, the artificial breeding of the long-spined sea star is particularly crucial for understanding the early development process of the long-spined sea star in the South China Sea and explaining the outbreak causes. SUMMARY

[0004] The purpose of the present application is to provide a preparation method for efficiently obtaining solar acanthaster larva and juvenile / larva and application thereof.

[0005] A preparation method for solar acanthaster larva and juvenile / larva, comprising the following steps: distinguishing the gonad development period and male and female gender of the solar acanthaster; injecting oxytocin into the male and female individuals, separately collecting the ovum of the female parent and the sperm of the male parent, adding the active sperm in batches after the ovum breaks, and the quantity ratio of the sperm and the ovum is 50-100:1; controlling the seawater temperature to be 28-30℃, the salinity to be 33-35 ppt, and the pH to be 8.11-8.17 during the hatching of the larvae, and culturing under full or half light shielding, and stirring when the fertilized eggs appear to be clustered; feeding the hatched larvae with feed algae, changing water and removing the dead and abnormal individuals; increasing the amount of feed after the development of the larvae to the brachiolaria, until more than 70% of the brachiolaria develops to the late brachiolaria with the size of 900-1100 μm x 550-650 μm and the body surface color of golden yellow, and then adding the attachment base until the 5-brachiolaria juvenile bodies are grown; washing the juvenile bodies from the attachment base in the intermediate growth stage, and transferring them to the environment containing live stones for culture until the size of the larvae is developed; and the culture conditions of the larvae, the juvenile bodies and the larvae are all seawater with the temperature of 28-31℃, the salinity of 33-35 ppt, and the pH of 8.11-8.17.

[0006] Preferably, the arm-bearing larvae develop to 900-1100 μm x 550-650 μm, the arm-bearing larvae develop to 1000 μm x 600 μm, the more than 70% is 70%, the attachment base is exposed to the sun and dead coral limb, and the live stone is covered with coral algae.

[0007] Preferably, the oxytocin is 1-methyladenine, the concentration is 1 mM, and the dosage is 10-15 mL.

[0008] Preferably, the dosage is 10 mL.

[0009] Preferably, the density of fertilized eggs during the hatching of larvae is 2 / mL.

[0010] Preferably, the concentration of feeding bait algae is 3000-4000 / mL, the bait algae is Nitzschina closterium, and the bait is filtered through a 200-mesh screen before feeding.

[0011] Preferably, the concentration of feeding bait algae is 3000 / mL, and the bait algae is Nitzschina closterium.

[0012] Preferably, the seawater for cultivation is taken from a coral reef area and filtered through a 10-μm screen.

[0013] Preferably, the gonadal development period of the sun long-spined sea star is as follows: if the gonad is golden yellow and full, or there are eggs with a diameter of 200 μm, it is the female mature period; if the gonad is white and full, it is the male mature period.

[0014] The application also provides the use of the preparation method in the feeding of sun long-spined sea star larvae, the cultivation of juvenile intermediates, the monitoring of larval natural enemies, or the early warning monitoring of larvae.

[0015] Advantages of the application:

[0016] The entire early life history of Solaria sunflower is described herein, and the use of 1-methyladenine to catalyze the production of a large number of gametes, combined with the advantages of artificial breeding, significantly improves the fertilization rate and hatching rate of the offspring of the sunflower sea star, and under suitable environmental conditions controlled by humans, bypasses the adverse factors in the natural environment, such as natural enemies and nutritional deficiencies, and then under the conditions of high-quality feed such as Chaetoceros muelleri, small new moon rhombus and suitable attachment base, the survival rate of the planktonic larvae is significantly improved to about 80%, and the metamorphosis rate is about 40%, the breeding cost and culture period are reduced, and a batch of larvae and juveniles can be obtained within 20 days, and a batch of juveniles can be obtained after 4 months. The invention helps the investigation, monitoring, early warning, disaster prevention and control, and disaster disposal of the sunflower sea star larvae / body, and plays a promoting role in the ecological safety of the island reefs.

[0017] Previously, the South China Sea Institute of the Chinese Academy of Sciences cultured 5-arm Pacific sunflower sea star complex juveniles in 2017 (Tian et al 2017), compared with the present invention, the present invention is specifically aimed at the sunflower sea star group that broke out in China's island reefs, and the present invention aims to provide a method for efficiently and quickly obtaining larvae and juveniles, which differs in terms of feed feeding and laboratory culture conditions, etc. Finally, the metamorphosis rate of 2.5% in the text is increased to about 40% now, which improves the breeding efficiency and reduces the cost.

[0018] The present invention can obtain a batch of sunflower sea star planktonic larvae and juveniles on a large scale, significantly improve the metamorphosis rate of the planktonic larvae, accelerate the growth rate and survival rate of the planktonic larvae, and obtain the juveniles to study the benthic attachment and metamorphosis process of the sunflower sea star, explore the process of the juveniles' food change, provide a new idea for the monitoring, early warning and prevention and control of the sunflower sea star, and can be widely used in the research fields of sunflower sea star larvae feeding, juvenile intermediate cultivation, juvenile natural enemies and larvae early warning monitoring, and has an important basic role in clarifying the outbreak of the sunflower sea star. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the fertilization and development process of the sunflower sea star, wherein (1) female gonad (2) male gonad (3) oogenesis bubble unbroken (4) oogenesis bubble broken (5) pinnularia (6) brachiolaria (7) juvenile (8) larva. DETAILED DESCRIPTION

[0020] The following examples are further illustrations of the present invention, but are not limitations of the present invention.

[0021] Example 1:

[0022] a. Sex identification: In May 2023, when it was summer in the South China Sea islands and reefs, the breeding season of long-spined sea star, 40 mature long-spined sea stars were collected into a culture tank (1.3 m x 0.8 m x 0.6 m) for temporary culture. The water for culture was pumped from the coral reef sea area to the laboratory by a 2200W submersible pump, and then filtered by a 10μm screen roller to be used for experimental culture. With the help of a 5mL syringe, the sex gland development period and male and female sex of long-spined sea star were distinguished. The 5mL syringe was inserted into the base of the long-spined sea star arm, and 1mL of sex gland was extracted and placed under a light microscope for observation. If there are 200μm diameter eggs, it can be judged as a female individual that can be used for breeding. The sex gland can also be used to determine if it is a mature female, if it is a full sex gland with a golden yellow color, it can be judged as a mature female, if it is a full sex gland with a white color, it can be judged as a mature male.

[0023] b. Artificial induction and fertilization: 3 females and 2 males were placed in separate culture tanks. A 1mM 1-methyl-adenine (purchased from MACKLIN company) inducer was prepared with a 2% NaCl solution. 10mL of freshly prepared inducer was injected into the base of the arms of the female and male individuals. After about 30 minutes, the eggs of the female and the sperm of the male were collected separately. Finally, 3 tanks (female tanks) of eggs and 2 small barrels of sperm were collected, and the 2 small barrels of sperm were mixed evenly. Before fertilization, check if the egg bubble has broken. When the bubble breaks, add the active sperm identified by microscopy to the three female tanks, control the ratio of sperm and eggs to be 50-100:1, and then according to the fertilization microscopy results of the eggs (whether the primary egg membrane appears), supplement the sperm in the form of "multiple small supplements" as needed until all the eggs in the field are fertilized. To prevent sperm contamination, it is customary to wash hands and experimental tools with fresh water before each operation. Figure 1 ) until all the eggs in the field are fertilized. To prevent sperm contamination, it is customary to wash hands and experimental tools with fresh water before each operation.

[0024] c. Larval hatching: The water temperature of the culture tank was controlled between 28-30℃, the salinity was between 33-35ppt, the pH was between 8.11-8.17, and the whole process was kept under micro-aeration and full / half light culture. During the hatching period, if the density of fertilized eggs is too large, they will cluster and attach to the bottom of the tank, which needs to be stirred frequently to improve the hatching success rate. During the hatching period, the density of fertilized eggs was maintained at 2 / mL.

[0025] d. Larvae culture: The larvae were selected by rolling net with 150 mesh screen and then were distributed into multiple polypropylene boxes (0.5 m x 0.4 m x 0.3 m) for culture, with a density of 1 larvae / mL. The same micro-aeration culture was continued, and the culture conditions were kept the same as in step c. The seawater in the tank was changed every three days to remove dead and abnormal individuals. When the seawater was changed, the larvae were first collected by rolling net with 150 mesh screen and then were transferred into the culture tank with fresh seawater. During the culture, the larvae were fed with 3000 small new moon-shaped algae / mL every day, with feeding twice a day. To ensure that no impurities were mixed in the culture process, the bait was filtered by rolling net with 200 mesh screen before feeding to remove algal powder residues and algal secretions. During the culture, floating impurities were removed by rolling net with 200 mesh screen.

[0026] e. Benthic attachment: The same culture conditions were maintained. When the culture reached the 12th day, the planktonic larvae developed into brachiolaria larvae, with a size of about 800 μm x 530 μm. At this time, the feeding amount was increased to 4000 larvae / mL, and the same morning and evening feeding was maintained. The culture was continued until the 20th day, until 70% of the larvae reached a size of 1000 μm x 600 μm. At this time, the larvae were golden yellow and visible to the naked eye, and multiple pairs of small arms were formed at the front end of the body. The brachiolaria larvae to be metamorphosed were collected by rolling net with 80 mesh screen and were transferred into a culture tank containing dead coral stumps (which had been treated by sun exposure for several days) for metamorphosis. At this time, the water was treated by the water purification method for 7-10 days, during which the water was not changed but aeration was maintained. Most of the larvae completed benthic metamorphosis, and the metamorphosed benthic larvae were golden yellow dots visible to the naked eye, with a size of 0.5 mm.

[0027] f. Intermediate culture: Suitable live rocks were selected and placed in new receiving boxes, and the live rocks were covered with a layer of benthic coral algae, without polychaetes and crustaceans. The coral stumps were washed by a water pick to make the juveniles fall off the attachment base, and the juveniles were collected by 150 mesh hand screen and were transferred into the tank containing live rocks for culture, with semi-flowing water. The culture conditions were the same as in step c.

[0028] The gender identification, fertilization and development process of the sun long-spined sea star in this example are shown in Figure 1 The early development process and development time of the sun long-spined sea star are shown in Table 1. Through the technical means of this example, 405 long-spined sea star juveniles were successfully bred after one month of culture, and the metamorphosis rate was increased to about 40%. After four months, 12 long-spined sea star larvae were obtained.

[0029] Table 1. Early development process and development time of the sun long-spined sea star

[0030]

[0031] Example 2:

[0032] a. Sex Identification: In May 2022, during the summer season on the South China Sea islands and reefs, which is also the breeding season for crown-of-thorns starfish, 30 healthy adult crown-of-thorns starfish were captured and temporarily held in culture tanks (1.3m × 0.8m × 0.6m). The culture water was pumped from the coral reef waters to the laboratory using a 2200W submersible pump and filtered through a 10μm sieve before being used for experimental culture. The gonadal development stage and sex of the crown-of-thorns starfish were identified using a 5mL syringe. Individuals with mature gonads were selected for group breeding. Egg diameter and gonad color were used to determine their suitability for breeding. A 5mL syringe was inserted into the base of the crown-of-thorns starfish's arm to extract 1mL of gonad, which was then observed under an optical microscope to assess gonadal development. Finally, 3 females and 3 males were selected for artificial breeding.

[0033] b. Artificial spawning and fertilization: Three female individuals were placed separately in white culture tanks, and three male individuals were placed in a 0.5m × 0.4m × 0.3m storage box. A 1mM 1-methyl-adenine (purchased from MACKLIN) spawning induction agent was prepared using a 2% NaCl solution. 15mL of the freshly prepared spawning induction agent was injected into the base of the brachial appendages of both male and female individuals. Approximately 30 minutes later, the parents began to produce sperm and release eggs. Finally, eggs from three tanks and sperm from three small containers were collected. The sperm from the three small containers were mixed thoroughly. Before fertilization, the follicles of the eggs were checked for rupture. After follicle rupture, sperm that were identified as active under a microscope were added to the three female tanks. Initially, a small amount of sperm was added at a sperm-to-egg ratio of 50-100:1. Subsequently, based on the results of fertilization microscopy (whether the primary egg membrane appeared), sperm was added in small increments as needed. Figure 1 This continues until all eggs in the field of view are fertilized. To prevent sperm contamination, hands and laboratory tools are habitually washed with fresh water before each step of the procedure.

[0034] c. Larval Hatching: During hatching, control the seawater temperature in the rearing tank between 28℃ and 30℃, the salinity between 33 and 35 ppt, and the pH between 8.11 and 8.17, maintaining slight aeration throughout the process, and culturing in full / semi-shade. If the density of fertilized eggs in the culture tank is too high, causing them to clump together and adhere to the bottom of the tank, frequent stirring is necessary to improve the hatching success rate. During hatching, maintain a fertilized egg density of 2 eggs / mL.

[0035] d. Larvae culture: The larvae were collected by 150 mesh screen and then distributed into polypropylene boxes (0.5 m x 0.4 m x 0.3 m) for culture, with a density of 1 larva / mL. The culture conditions were the same as those in step c. The seawater in the culture tank was changed every three days to remove dead and abnormal individuals. The larvae were collected by 150 mesh screen and then transferred into a new box with fresh seawater. During the culture, the larvae were fed with 3000 small new moon-shaped algae / mL every day, twice a day. To ensure that no impurities were mixed during the culture, the bait was filtered by a 200 mesh screen before feeding to remove residues and algal secretions. During the culture, floating impurities were removed by a 200 mesh screen.

[0036] e. Benthic attachment: The culture conditions were kept the same. When the larvae developed into brachiolaria larvae at the 12th day, the larvae grew to about 800 pm x 530 pm. After 17 days, the brachiolaria larvae developed into post-metamorphic brachiolaria larvae. At this time, the larvae were golden yellow and the front end of the body formed multiple small arms. The post-metamorphic brachiolaria larvae were collected by an 80 mesh screen and then transferred into a culture tank containing coral stones for metamorphosis. The water was not changed during the culture, but aeration was maintained. After 7 days, the larvae completed the benthic metamorphosis stage, and the metamorphosed benthic larvae attached to the coral stones and were golden yellow dots with a size of 0.5 mm.

[0037] f. Intermediate growth: The suitable live stones were selected and placed in a new box. The live stones were covered with a layer of benthic coral algae, and no polychaetes and crustaceans were present. The coral stones were washed by a water pick to make the juveniles fall off the attachment base. The juveniles were collected by a 150 mesh screen and then transferred into a tank containing live stones for semi-aquatic culture. The culture conditions were the same as those in step c.

[0038] Through the technical means of this embodiment, we successfully bred 20 long-spined sea star juveniles with a metamorphosis rate of about 2% after one month of culture. After four months, we obtained 0 long-spined sea star larvae.

Claims

1. A method for preparing larvae and juveniles / larvae of the sun-borne starfish, characterized in that, Includes the following steps: Identify the gonadal development stage and sex of the sun crown-of-thorns starfish; inject oxytocin into male and female individuals, collect eggs from the female and sperm from the male separately, and add active sperm in stages after the eggs rupture, with a sperm-to-egg ratio of 50-100:1; during larval hatching, control the seawater temperature at 28℃-31℃, salinity at 33-35 ppt, and pH at... From August 11th to 17th, the larvae are cultured in full or partial shade, and stirred when fertilized eggs cluster together. After hatching, the larvae are fed algae, the water is changed, and dead and deformed individuals are removed. Once they develop into arm-like larvae, the amount of food is increased. When more than 70% of the arm-like larvae have developed to a size of 900-1100μm × 550-650μm, an attachment substrate is added until they grow into 5-armed juveniles. Then, they are transferred to an environment containing live rock for further cultivation, eventually developing into larvae. The culture conditions for larvae, juveniles, and larvae are: seawater temperature of 28℃-31℃, salinity of 33-35ppt, and pH of 8.11-8.

17. The attachment substrate is sun-exposed dead coral fragments, and the live rock is coral stone covered with coral algae.

2. The preparation method according to claim 1, characterized in that, The oxytocin mentioned is 1-methyladenine, with a concentration of 1 mM and a dosage of 10-15 mL.

3. The preparation method according to claim 2, characterized in that, The dosage is 10 mL.

4. The preparation method according to claim 1, characterized in that, The density of fertilized eggs during the larval hatching period is 2 eggs / mL.

5. The preparation method according to claim 1, characterized in that, The concentration of algae in the feed is 3000-4000 cells / mL. The algae used for the feed is *Neptune pulvinatum*. Before feeding, the feed is filtered through a 200-mesh silk screen to remove residues and algal secretions.

6. The preparation method according to claim 1 or 5, characterized in that, The concentration of the algae used as bait is 3000 algae / mL, and the algae used as bait is *Nyctaginosa*.

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