A method for breeding Chinese horseshoe crabs

Abstract

The application discloses a Chinese horseshoe crab breeding method and belongs to the technical field of aquaculture. The Chinese horseshoe crab breeding method is characterized by the following steps: selecting Chinese horseshoe crab seedlings and placing the seedlings in a culture box, and laying a breeding substrate at the bottom of the culture box, wherein the breeding substrate comprises sediments collected from a natural habitat of the Chinese horseshoe crab. The breeding method can promote the development of Chinese horseshoe crab seedlings, especially the growth and development of diapause 1-year-old Chinese horseshoe crabs, and improve the molting rate and survival rate of the Chinese horseshoe crabs.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method and application for cultivating Chinese horseshoe crab seedlings, especially diapause-aged Chinese horseshoe crabs, by utilizing habitat enrichment. Background Technology

[0002] The Chinese horseshoe crab (Tachypleus tridentatus) is a large, benthic arthropod. Its blue blood is an important raw material for preparing horseshoe crab reagents for pyrogen detection. The Chinese horseshoe crab is distributed throughout Asia, and my country accounts for over 90% of the global population. Over the past three decades, numerous studies have shown that the Chinese horseshoe crab population has experienced a sharp decline, leading to its assessment as "endangered" by the IUCN in 2019 and its inclusion in my country's list of Class II protected wild animals in 2021.

[0003] With the natural population declining, it is crucial to enhance and restore its resources through artificial breeding. Artificial breeding of the Chinese horseshoe crab is still in its early stages, and the breeding technology is extremely immature. Domestic enterprises producing Chinese horseshoe crab seedlings are only scattered in Guangdong, Guangxi, and Fujian provinces, producing mainly one-year-old seedlings, with a severe shortage of larger seedlings of two years and above. From one-year-old larvae to sexual maturity, the Chinese horseshoe crab undergoes approximately 15-16 molts (12-13 years), with the individual growing and developing with each molt. An organism's life history consists of a series of active growth and reproductive periods; the phenomenon of these cycles being delayed or stopped due to specific environmental stimuli is called diapause. The inventor's team has been conducting research on the artificial breeding of Chinese horseshoe crabs for nearly ten years. Based on years of practice, they have found that artificially bred Chinese horseshoe crabs undergo a significant diapause process during periods of temperature fluctuation, especially during the low-temperature winter period (below 24℃). Typically, one-year-old larvae hatched in September of the same year do not molt until late April of the following year (up to 7 months), with a proportion exceeding 95%. Generally, when raised at 28℃-34℃, the time range for 1-year-old horseshoe crab larvae to develop into 2-year-old larvae is 60-90 days. It can be seen that diapause not only prolongs the breeding cycle of Chinese horseshoe crab larvae, but also leads to difficulties in molting (molting rate less than 5%), which in turn causes a surge in mortality (more than 90%).

[0004] To date, research on horseshoe crab diapause in China is almost nonexistent both domestically and internationally. The problems of difficult molting and development of horseshoe crab seedlings and soaring mortality rates have not received sufficient attention, leaving large-scale seedling production in a predicament of technological gaps. Summary of the Invention

[0005] In order to overcome the problem of low molting rate and high mortality rate of diapause horseshoe crab larvae, the present invention aims to provide a cultivation method that promotes the development of horseshoe crab seedlings, especially diapause horseshoe crabs, by adding substrate to enrich the habitat.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for cultivating horseshoe crabs (Heliotropium indicum), comprising the following steps: Chinese horseshoe crab seedlings were selected and placed in an incubator for cultivation. The bottom of the incubator was covered with a culture substrate, which contained sediments collected from the natural habitat of the Chinese horseshoe crab. The horseshoe crab seedlings mentioned are diapause 1-year-old horseshoe crabs and / or normal 1-2 year-old horseshoe crabs; wherein the diapause 1-year-old horseshoe crabs are juvenile horseshoe crabs that have not molted to 2 years old by April of the following year after initial hatching in September of the current year.

[0007] Existing methods for raising juvenile horseshoe crabs in China often employ a simple, extensive approach of stocking them in water without adding any substrate, failing to consider the horseshoe crab's habitat requirements. This invention, while mimicking nature, adds substrate from the horseshoe crab's natural habitat to the culture tank, providing a simulated natural environment for the juvenile seedlings. This satisfies the ecological habits of the Chinese horseshoe crab, allowing it to revert to its natural burrowing and sand-diving behaviors, thus promoting its growth and development.

[0008] In some embodiments of the present invention, the natural habitat of the Chinese horseshoe crab is the intertidal mudflat. The Chinese horseshoe crab is a typical marine benthic organism, and its life cycle requires key habitats including the high tide line as the spawning and breeding ground for adults, the intertidal mudflat as the juvenile rearing ground, and the subtidal zone (30m nearshore) as the habitat for adults. Therefore, in its natural state, juvenile horseshoe crabs mainly inhabit and forage in the intertidal mudflat. Collecting intertidal mudflat sediments is more in line with the natural living environment of juvenile horseshoe crabs and promotes seedling development.

[0009] In some embodiments of the present invention, the thickness of the aquaculture substrate is 2-5cm, such as 2cm, 3cm, 4cm, 5cm, etc.

[0010] In some embodiments of the invention, the sediment comprises muddy sediments and / or sandy sediments, and may be a single muddy sediment, sandy sediment, or a mixture of both. In some preferred embodiments, the aquaculture substrate is a mixture of muddy sediments and sandy sediments in a 1:1 weight ratio.

[0011] In some embodiments of the present invention, the particle size of the sandy sediment is 0.06-0.6 mm, and sandy sediments of suitable particle size can be obtained by sieving and natural drying. For example, the particle size of the sandy sediment is 0.1-0.6 mm, 0.06-0.5 mm, 0.1-0.5 mm, 0.3-0.6 mm, etc.

[0012] In some embodiments of the present invention, the culture water in the incubator is derived from seawater, and the physicochemical properties of the culture water meet at least one of the following requirements: water temperature 28-32℃, salinity 28-30 ppt, pH 7.5-8.0, dissolved oxygen 6.8-7.4 mg / L, ammonia nitrogen content ≤0.10 mg / L, and nitrite concentration ≤0.001 mg / L. The culture water can be obtained through natural seawater filtration, etc., and the physicochemical properties of the culture water are kept stable during the cultivation process to provide a stable environment for horseshoe crab growth.

[0013] In some embodiments of the present invention, the culture density in the incubator is controlled at 3000-5000 individuals / m². 3 This is to ensure that diapause juvenile horseshoe crabs have sufficient habitat space, which facilitates the restoration of their natural instincts and reduces stress or even damage caused by long-term stacking, thereby improving the growth performance and survival rate of diapause juvenile horseshoe crabs.

[0014] In some embodiments of the present invention, fresh oyster meat is used to feed juvenile horseshoe crabs during the cultivation process. Existing methods for cultivating Chinese horseshoe crab seedlings often lack proper daily management, leading to insufficient nutrient intake and diapause in juvenile horseshoe crabs. Preferably, the crude protein content of the oyster meat is ≥8 wt%. Preferably, the amount of oyster meat fed is 10-15% of the horseshoe crab's body weight. In some preferred embodiments, oyster meat with a crude protein content ≥8 wt% is used as feed, fed once daily at 10-15% of the juvenile horseshoe crab's body weight to meet the nutritional needs for the growth and development of diapause juvenile horseshoe crabs, thereby promoting molting and improving their survival rate.

[0015] In some embodiments of the present invention, the culture water in the culture box is changed every 2 days, with 80-90% of the original culture water volume replaced each time. By changing the water, the water quality can be kept clean to meet the survival needs of juvenile horseshoe crabs, further helping the juvenile horseshoe crabs adapt to the environment, thereby improving the molting rate and survival rate of juvenile horseshoe crabs, especially diapause juvenile horseshoe crabs.

[0016] In some embodiments of the present invention, during the cultivation process, the cultivation light cycle is maintained to simulate the natural living environment of juvenile horseshoe crabs and promote their development.

[0017] In some embodiments of the present invention, when Chinese horseshoe crab seedlings are placed in an incubator for cultivation, the transfer and cultivation operation is carried out after 17:30 in the afternoon to better facilitate the horseshoe crabs' adaptation to the environment.

[0018] Compared with the prior art, the present invention has the following advantages: 1) The method of this invention follows the principle of nature and cultivates Chinese horseshoe crab seedlings by adding substrate to create a habitat enrichment, especially 1-year-old diapause Chinese horseshoe crabs. It can improve the survival rate of diapause juvenile horseshoe crabs, the molting rate of 1-year-old diapause juvenile horseshoe crabs, and the survival rate of molted individuals, thus solving an industry problem.

[0019] 2) This invention cultivates horseshoe crabs by adding substrate to the aquaculture water. Firstly, the technical operation is simple and easy to understand, and does not require special instruments and equipment. Secondly, the substrate is easy to obtain, which makes it easy to train practitioners to use the substrate to create habitat enrichment for the cultivation of diapause juvenile horseshoe crabs. Furthermore, the operation process is simple and easy to replicate, making it easy to promote on a large scale.

[0020] 3) This invention can improve the molting rate of diapause juvenile horseshoe crabs and obtain high-quality, large-sized seedlings, which is beneficial to the development of China's horseshoe crab artificial breeding industry. Attached Figure Description

[0021] Figure 1 Add an image of diapause-stage 1-year-old Chinese horseshoe crabs cultured in sandy substrate to Example 1; Figure 2 Add an image of 1-year-old horseshoe crabs in diapause on a muddy substrate for Example 2; Figure 3 Image of diapause-aged Chinese horseshoe crabs cultured in sandy soil and sandy substrate (1:1) as shown in Example 3; Figure 4 Image of a 1-year-old Chinese horseshoe crab cultured without substrate (for Comparative Example 1); Figure 5 The image shows a diapause 1-year-old Chinese horseshoe crab molting into a 2-year-old after being cultivated in Example 3. Detailed Implementation

[0022] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] Unless otherwise specified, all other materials, reagents, etc. used in the examples and comparative examples are commercially available.

[0024] Explanation of the source of Chinese horseshoe crab seedlings: The 1-year-old diapause juvenile horseshoe crabs used in the following examples and comparative examples were provided by the Chinese Horseshoe Crab Artificial Breeding Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. Their initial weight was 0.026-0.028g and their carapace width was 0.5-0.6cm.

[0025] The breeding season for Chinese horseshoe crabs at this base is from April to September each year. The parent horseshoe crabs lay eggs in multiple batches, and all fertilized eggs are incubated in indoor cement tanks. They hatch into one-year-old juveniles in about 60 days. After hatching, the one-year-old juveniles are then raised in water. From September of the current year to April of the following year, drastic temperature fluctuations (sudden drops in temperature and large diurnal temperature variations) can induce diapause in the one-year-old juveniles. By late April of the following year, over 95% of the one-year-old juveniles remain in the diapause stage. This base can produce over 200,000 one-year-old diapause horseshoe crabs annually.

[0026] Example 1 This embodiment provides a breeding method for promoting the development of diapause-aged Chinese horseshoe crabs, comprising the following steps: (a) Preparation of incubator The incubator is a PVC water tank with dimensions of 1m×1m×1m. Several tanks are available as spares. Each incubator is equipped with an oxygen pump, and each oxygen pump is connected to two air stones via an air distribution valve and placed into each incubator.

[0027] Seven days before transferring the horseshoe crab seedlings, the inner wall of the incubator was thoroughly disinfected by evenly spraying a chlorine dioxide solution with a concentration of 2 mg / L.

[0028] (II) Substrate preparation Sandy sediments collected from the natural habitat of juvenile horseshoe crabs—the sandy tidal flats of Fangchenggang, Guangxi—were transported to the aquaculture base. The sediments were sieved to remove impurities such as dead leaves, large insects, etc., leaving sand with a particle size of 0.1-0.6 mm that was allowed to air-dry naturally. The dried sand was then added to the culture tanks, covering the bottom to a depth of 3 cm as the culture substrate.

[0029] (III) Preparation of aquaculture water Natural seawater is drawn and introduced into the water storage tank of the cultivation workshop (approximately 9m×8m×3m in size) through a pipeline. After 48 hours of dark sedimentation in the water storage tank, the seawater passes through a coarse sand tank, a fine sand tank, and a screen for filtration before being piped into the cultivation box with substrate obtained in step (II) to a height of 0.3m.

[0030] (iv) Seedling release Transfer the 1-year-old horseshoe crab seedlings to the incubator prepared in step (III) for further cultivation. The transfer operation should be carried out after 17:30 in the afternoon.

[0031] (v) Seedling cultivation 5.1 Cultivation Density Management The density of juvenile horseshoe crabs was controlled at 4000 individuals / m² throughout the entire breeding process. 3 .

[0032] 5.2 Water Quality Management During the cultivation period, the water temperature is maintained at 30±1℃, the salinity at 28-30ppt, the pH at 7.5-8.0, the dissolved oxygen at 6.8-7.4mg / L, the ammonia nitrogen at less than 0.10mg / L, and the nitrite concentration at less than 0.001mg / L.

[0033] 5.3 Feeding Management Throughout the rearing process, oyster meat is used as feed. Fresh oyster meat is ground using a grinder (CG-8316, Long Plus), filtered through a 200-mesh screen, and the resulting meat paste is used as feed. The oysters are fed once daily at 8:00 AM at a rate of 10% of their body weight.

[0034] 5.4 Water Change and Sludge Removal Throughout the rearing process, the water is changed every two days. First, use a sponge to wipe away slime and other debris from around the rearing tank, while thoroughly stirring the sand at the bottom to expose any uneaten food. Then, use a siphon with a filter membrane (to prevent backflow of juvenile horseshoe crabs and sand) to siphon out all the debris from the tank. This process replaces 90% of the original seawater. Finally, add the fresh seawater prepared in step (II) to the set level to complete the water change. Depending on the bottom sediment depth of 3cm, supplement the substrate as shown in step (IV) as needed.

[0035] Example 2 The difference from Example 1 is that in step (ii) above, the substrate added to each incubator is 100% muddy sediment, which is collected from the muddy tidal flats of Naozhou Island in Leizhou Bay and has a particle size range of 0.1-0.6 mm.

[0036] Example 3 The difference from Example 1 is that in step (ii) above, the substrate added to each incubator is a mixture of muddy and sandy sediments, wherein the sandy sediments have a particle size range of 0.06-0.5 mm, the weight ratio of muddy and sandy sediments is 1:1, and both muddy and sandy sediments are collected from the muddy and sandy tidal flats of Suixi, Zhanjiang, Beibu Gulf.

[0037] Comparative Example 1 The difference from Example 1 is that step (II) is omitted and no substrate is added.

[0038] 48,000 one-year-old diapause horseshoe crabs were randomly divided into four groups and cultured according to the methods of Example 1, Example 2, Example 3 and Comparative Example 1, respectively. Each group had three replicates, with 4,000 juvenile horseshoe crabs in each replicate. The experiment lasted for 35 days.

[0039] During the experiment, the molted shells of juvenile horseshoe crabs were collected daily, and the number of molted shells, deaths, and survivals were recorded. After the experiment, the cumulative molting rate, total survival rate, and survival rate of molted individuals (i.e., survival rate of 2-year-old individuals) were calculated.

[0040] Overall survival rate (%) = 100 × S t / S0; Cumulative molting rate (%) = (M i / C r )×100; Survival rate of molted individuals (%) = (S Mt / M it )×100; In the formula: S t S0 represents the number of surviving horseshoe crabs at the end and the beginning; M i The cumulative number of individuals that molted at the end; C rS represents the total number of individuals in the group at the beginning. Mt M represents the number of molting individuals that survived within the group at the end of the molting process. it The total number of individuals molting within the group at the end of the term.

[0041] SPSS 22.0 was used to perform statistical analysis on the above three indicators. After the original data were normalized and tested for homogeneity of variance, one-way ANOVA and Duncan's post-hoc multiple comparisons were performed. The significance level was set at p<0.05.

[0042] Figure 1-3 The images are from Examples 1-3, showing the cultivation of diapause-aged Chinese horseshoe crabs with different substrates. Figure 4 The image shows a 1-year-old Chinese horseshoe crab cultured without substrate in Comparative Example 1. Figure 5 The image shows horseshoe crabs cultured in Example 3 that have molted from one-year-old diapause stage to two-year-old stage. The survival rate and molting rate statistics for each example and comparative example are shown in Table 1.

[0043] Table 1. Statistical results of survival rate and molting rate of the examples and comparative examples.

[0044] Note: The same lowercase letter after each row of values ​​indicates that the difference between groups is not significant (p>0.05), while different lowercase letters indicate that the difference between groups is significant (p<0.05).

[0045] Based on the above experimental results, compared with the existing substrate-free cultivation methods, the cultivation method of this invention can significantly promote the growth of diapause-aged horseshoe crabs and significantly improve the molting rate of diapause-aged horseshoe crabs and the survival rate of molting-aged horseshoe crabs. In particular, the cultivation effect is better with a 1:1 mixture of mud and sand substrate, which can increase the total survival rate from 31.33% to 63.17%, the survival rate of molting-aged individuals from 20.95% to 79.11%, and the cumulative molting rate from 2.67% to 50.67%.

[0046] It should be noted that although the above embodiments only use 1-year-old horseshoe crabs as an example to illustrate the cultivation method of the present invention, it is not difficult for those skilled in the art to realize that the cultivation method of this application can also be used for the cultivation of normal 1-2 year-old horseshoe crab seedlings.

[0047] For those skilled in the art, several simple deductions or substitutions can be made without departing from the inventive concept, without requiring creative effort. Therefore, any simple improvements made to this invention by those skilled in the art based on the disclosure of this invention should be within the scope of protection of this invention. The above embodiments are preferred embodiments of this invention, and all processes similar to this invention and equivalent changes should fall within the scope of protection of this invention.

Claims

1. A method for cultivating horseshoe crabs (Heliotropium indicum), characterized in that, Includes the following steps: Chinese horseshoe crab seedlings were selected and placed in an incubator for cultivation. The bottom of the incubator was covered with a culture substrate, which contained sediments collected from the natural habitat of the Chinese horseshoe crab. The horseshoe crab seedlings mentioned are diapause 1-year-old horseshoe crabs and / or normal 1-2 year-old horseshoe crabs; wherein the diapause 1-year-old horseshoe crabs are juvenile horseshoe crabs that have not molted to 2 years old by April of the following year after initial hatching in September of the current year.

2. The cultivation method according to claim 1, characterized in that, The natural habitat of the Chinese horseshoe crab is an intertidal mudflat. And / or, the sediments include muddy sediments and / or sandy sediments; And / or, the thickness of the aquaculture substrate is 2-5cm.

3. The cultivation method according to claim 2, characterized in that, The sandy sediments have a particle size of 0.06-0.6 mm.

4. The cultivation method according to claim 1, characterized in that, The culture water in the incubator is sourced from seawater, and the physicochemical properties of the culture water satisfy at least one of the following: Water temperature 28-32℃ Salinity 28-30 ppt pH 7.5-8.0, Dissolved oxygen 6.8-7.4 mg / L Ammonia nitrogen content ≤0.10mg / L, Nitrite concentration ≤ 0.001 mg / L.

5. The cultivation method according to claim 1, characterized in that, The incubation density in the incubator is controlled at 3000-5000 individuals / m². 3 .

6. The cultivation method according to claim 1, characterized in that, During the cultivation process, fresh oyster meat is used as feed.

7. The cultivation method according to claim 6, characterized in that, The crude protein content of the oyster meat paste is ≥8wt%; and / or the amount of oyster meat paste fed is 10-15% of the weight of the horseshoe crab.

8. The cultivation method according to claim 1, characterized in that, The culture water in the incubator is changed every 2 days, with 80-90% of the original culture water volume replaced each time.

9. The cultivation method according to claim 1, characterized in that, During the cultivation process, the cultivation light cycle is kept in accordance with the natural light cycle.

10. The cultivation method according to any one of claims 1-9, characterized in that, When placing Chinese horseshoe crab seedlings in an incubator for cultivation, the transfer and cultivation operation should be carried out after 17:30 in the afternoon.

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