A breeding method to improve the molting rate and survival rate of diapause-aged Chinese horseshoe crabs.

Abstract

The present invention discloses a cultivation method for improving the molting rate and survival rate of diapause first-instar Chinese horseshoe crabs. The diapause first-instar Chinese horseshoe crabs refer to individuals that are hatched into first-instar juveniles in September of the current year and have not molted into second-instar horseshoe crabs until April of the following year. The method comprises the following steps: selecting diapause first-instar Chinese horseshoe crabs and placing them in seawater containing 30-90 mg / L of vitamin C for cultivation. The present invention can not only effectively improve the molting rate of diapause first-instar juvenile horseshoe crabs but also improve the survival rate of the juvenile horseshoe crabs, thereby obtaining second-instar horseshoe crab seedlings with good quality.

Description

Technical Field

[0001] This invention relates to the field of Chinese horseshoe crab seedling cultivation technology, specifically to a cultivation method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs. Background Technology

[0002] The Chinese horseshoe crab (a marine arthropod) Tachypleus tridentatus ) belongs to the class Mesostomata ( Xiphosura ), Stegostomata ( Xiphosurida The horseshoe crab (Heliotropium indicum) has survived for over 480 million years and still retains its original form, thus earning the reputation of a "living fossil." It is mainly distributed along the southeastern coast of my country and in Southeast Asian countries. Before the 1970s, my country's horseshoe crab resources accounted for over 95% of the world's total, especially in Fujian, Guangdong, and the Beibu Gulf region of Guangxi.

[0003] The blue blood of the Chinese horseshoe crab is the primary raw material for the global production of horseshoe crab reagents. These reagents are highly sensitive in detecting endotoxins produced by bacteria and are used globally in endotoxin testing for medical devices, pharmaceuticals, blood products, and vaccines. Therefore, horseshoe crab blood is irreplaceable in safeguarding global human health and the development of the healthcare industry, comparable to a strategic resource. However, numerous studies show that the Chinese horseshoe crab population has experienced a sharp decline over the past three decades. The IUCN assessed the Chinese horseshoe crab as "endangered" in 2019, and my country listed it as a Class II protected wild animal in 2021. Therefore, artificial breeding and stock enhancement are currently the most ideal and strategically significant measures for protecting horseshoe crab resources. Traditional stock enhancement typically uses artificially bred one-year-old juveniles, but the survival rate of one-year-old juveniles in the wild is extremely low, less than 10%, resulting in unsatisfactory resource restoration effects. Stock enhancement using two-year-old or older juveniles can improve the restoration effect of horseshoe crab resources.

[0004] Currently, only a small number of enterprises in Guangdong, Guangxi, and Fujian provinces in China produce horseshoe crab seedlings. These are all small-scale farms, producing mainly one-year-old larvae, with a severe shortage of larger larvae aged two years and above. Based on the inventor's team's nearly 10 years of artificial breeding practice of horseshoe crabs and visits to various horseshoe crab seedling production enterprises, it was found that diapause is a common phenomenon in the seedling cultivation of horseshoe crabs in China. Diapause refers to a phenomenon where the life cycle is delayed or stopped. From one-year-old larvae to sexual maturity, horseshoe crabs undergo 15-16 molts (approximately 12-13 years). Each molt signifies one growth cycle, making molting a crucial stage in the life cycle of horseshoe crabs. Generally, under conditions of 24-32℃, the development time for 1-4 year old larvae is 60-90 days. However, when horseshoe crabs in China experience diapause, the main characteristics are: they do not molt for a long time (far exceeding the 60 to 90 days that the normal development of 1-4 year old larvae is believed to take), their weight and the width of their carapace hardly change, and without care measures, the vast majority (>95%) of them fail to molt and eventually die.

[0005] The inventors' team investigated horseshoe crab seedling production enterprises in different regions from 2023 to 2025 and found that these enterprises used high-density farming methods (such as...) Figure 1 As shown in the figure, the diapause rate of 1-year-old horseshoe crabs is as follows: 90%~95% in Zhanjiang, Guangdong; 92%~93% in Yangjiang, Guangdong; 95%~96% in Shantou, Guangdong; and 90%~95% in Beihai, Guangxi. In these enterprises, diapause 1-year-old juvenile horseshoe crabs remain in diapause for 6~7 months without molting. Typically, fertilized eggs hatch into 1-year-old juveniles in September of the same year, but fail to molt into 2-year-old juveniles by April of the following year. Their weight and carapace width are almost unchanged compared to newly hatched 1-year-old juveniles (carapace width of 0.5~0.6 cm and weight of approximately 0.02g). These diapause juvenile horseshoe crabs often have a molting rate of less than 5% in subsequent aquaculture, and their mortality rate exceeds 90% (e.g., ...). Figure 2 The image shows a 1-year-old diapause juvenile horseshoe crab. Chinese patent publication CN102487853A discloses temperature as a key factor in the development of 1-year-old horseshoe crabs into 2-year-old juveniles; however, even with temperature control, only 60% of 1-year-old horseshoe crabs develop into 2-year-old juveniles, meaning that 40% of individuals still fail to molt to 2-year-old age. It is evident that current technology lacks measures to address the large-scale mortality of diapause 1-year-old Chinese horseshoe crabs.

[0006] Furthermore, traditional theory holds that juvenile horseshoe crabs can survive on endogenous nutrition during their first year of life. Therefore, in actual seedling production, they are usually not fed during this period. For example, Chinese patent CN102487853A discloses that juvenile horseshoe crabs are not fed during the rearing period from first to second year, with the first-year-old juveniles absorbing the yolk stored in their embryos as the nutrients needed for development. However, this no-feed rearing method, over time, can easily lead to insufficient nutrient reserves in the horseshoe crabs and weaken their environmental adaptability. The seedling rearing results under this management method are often unsatisfactory. The most prominent characteristic is that most first-year-old juvenile horseshoe crabs fail to molt and develop normally, resulting in near-zero growth. Coupled with seasonal temperature fluctuations and the lack of corresponding management equipment or measures in production, this leads to a large number of seedlings stagnating.

[0007] As mentioned earlier, diapause leads to the failure and death of a large number of 1-year-old juveniles, hindering the cultivation of large-sized horseshoe crab juveniles. Since the recovery of horseshoe crab populations, primarily through stock enhancement, depends on large-scale seed production by enterprises to provide high-quality seedlings, there is an urgent need to develop technologies that promote diapause molting and improve the survival rate of horseshoe crabs, thereby enhancing the effectiveness of horseshoe crab juvenile cultivation. Summary of the Invention

[0008] The purpose of this invention is to provide a breeding method for improving the molting rate and survival rate of 1-year-old Chinese horseshoe crabs, thereby promoting molting and increasing their survival rate.

[0009] To achieve the first objective mentioned above, the present invention adopts the following technical solution.

[0010] A breeding method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs, characterized by comprising the following steps:

[0011] One-year-old horseshoe crabs were selected and cultured in seawater containing 30-90 mg / L of vitamin C.

[0012] The diapause-stage 1-year-old Chinese horseshoe crabs described in this invention refer to individuals that hatch into 1-year-old juveniles in September of the current year and have not yet molted into 2-year-old horseshoe crabs by April of the following year.

[0013] This invention is the first to propose cultivating horseshoe crabs in diapause by adding water-soluble vitamin C to the water. The addition of vitamin C can alleviate the stress response induced by long-term diapause in juvenile horseshoe crabs, while enhancing their immunity, thereby improving their growth performance, promoting molting, and increasing their survival rate.

[0014] Preferably, the breeding density of Chinese horseshoe crabs is controlled at 5-8 individuals / m². 3 This ensures that juvenile horseshoe crabs have enough living space to swim, crawl, and forage, while minimizing the stacking of individual juveniles to avoid oxygen deprivation or fungal growth caused by stacking, thus providing a suitable growth environment for juvenile horseshoe crabs to promote molting.

[0015] Preferably, during the rearing process, the aquaculture water is changed every 2 days to ensure the water quality conditions required for the growth and development of juvenile horseshoe crabs and reduce the problem of increased ammonia nitrogen and nitrite content caused by pollution from uneaten feed, feces, etc. The specific operation for changing the water is as follows: first discard 90% of the volume of aquaculture water, and then add seawater containing 30~90mg / L vitamin C to the original volume.

[0016] Preferably, during the rearing process, fresh oyster meat is used to feed the juvenile horseshoe crabs; specifically, fresh oyster meat is fed once a day at a rate of 10% of the juvenile horseshoe crab's body weight. The crude protein content of the oyster meat is ≥8%, and as feed for the diapause juvenile horseshoe crabs, regular and quantitative feeding ensures the necessary nutrients for their growth and development. More preferably, the weight changes of the juvenile horseshoe crabs are sampled and monitored every two weeks, and the feeding amount is adjusted to 10% of their body weight based on the weight changes.

[0017] Preferably, the temperature of the aquaculture water is maintained at 24~32℃ during the cultivation process.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention improves the molting rate and survival rate of diapause-aged horseshoe crabs: By adding 30-90 mg / L of vitamin C to the water to cultivate diapause-aged horseshoe crabs, this invention increases the molting rate by 17.07%-52.05%, and the total survival rate (including 1-year and 2-year-old horseshoe crabs) by 38.67%-68.33%, of which the survival rate of molted 2-year-old horseshoe crabs reaches 69.54%-91.68%. Therefore, this invention uses vitamin C to cultivate diapause-aged horseshoe crabs, which can obtain more large-sized seedlings, and at the same time makes the sustainable cultivation of larger-sized seedlings feasible.

[0020] (2) This invention can improve aquaculture efficiency: This invention uses vitamin C to improve the molting rate and survival rate of diapause-aged horseshoe crabs, and can obtain high-quality 2-year-old horseshoe crab seedlings from a large number of diapause-aged seedlings without the need for special equipment and facilities; moreover, the market supply of vitamin C products is stable and the price is affordable (0.18 yuan / g-0.20 yuan / g), and it is simple and convenient to use. Therefore, this invention uses mature and economical vitamin C to carry out the aquaculture of diapause-aged horseshoe crabs, which can significantly improve aquaculture efficiency.

[0021] (3) This invention has strong applicability and scalability: This invention uses water-soluble vitamin C, which can be directly added to the aquaculture water to cultivate diapause horseshoe crabs. The operation is simple and easy to understand, and does not require special instruments and equipment, which can save a large number of diapause seedlings. Its low cost, convenient and effective operation make it easy to promote in actual production, especially in large-scale aquaculture enterprises, which is conducive to the development of horseshoe crab artificial aquaculture. Secondly, this technology can be promoted as a convenient and effective management method for the healthy cultivation of horseshoe crab seedlings to control the risks caused by diapause of seedlings at different developmental stages and promote the sustainable and large-scale production of larger-sized seedlings. Given the urgency of the protection of horseshoe crab biological resources, the supply of large-sized seedlings is a prerequisite for carrying out stock enhancement and release to achieve resource conservation. This invention is worthy of promotion and easy to promote at the level of horseshoe crab seedling cultivation technology. Attached Figure Description

[0022] Figure 1 This indicates that the traditional high-density farming methods used by horseshoe crab seedling companies in China have resulted in overcrowding.

[0023] Figure 2 The image shows 1-year-old horseshoe crabs in diapause. The red arrows point to 1-year-old horseshoe crabs that failed to molt into 2-year-old larvae during diapause; some of them are already dead or near death.

[0024] Figure 3 The example shown is that 1-year-old horseshoe crabs in diapause molted after being cultured with added vitamin C. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0026] Example 1

[0027] The breeding method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs in this embodiment includes the following steps:

[0028] (a) Preparation of indoor seedling trays

[0029] Preparation of indoor nursery ponds: The indoor nursery ponds are cement ponds, with dimensions of 8m long × 5m wide × 1.5m high. Several ponds are prepared for backup and are located in the horseshoe crab nursery. Each nursery pond is equipped with an aerator pump, and each aerator pump is connected to 10 air stones via an air distribution valve and placed into each nursery pond. Seven days before transferring the horseshoe crab larvae, the nursery ponds are thoroughly disinfected by evenly sprinkling a 2mg / L chlorine dioxide solution.

[0030] (II) Preparation of water for seedling raising

[0031] Fresh seawater is pumped from the offshore area and piped into the workshop's storage tank (approximately 9m × 8m × 3m). In the tank, the seawater undergoes 24 hours of dark sedimentation, followed by disinfection with 5 ppm chlorine dioxide. After approximately 24 hours, it is neutralized with 2.5 ppm sodium thiosulfate, and finally irradiated with ultraviolet (UV) lamps for about 1 hour. The UV lamps are approximately 30W with a wavelength of 200–270 nm, suspended vertically about 2 meters above the ground. The treated seawater is aerated for over 37 days and undergoes a three-stage sand filtration process: the first stage involves filtering small stones, a 20-mesh sieve, and coarse sand; the second stage involves filtering fine sand through an 80-mesh sieve; and the final stage involves filtering fine sand through activated carbon and a 150-mesh sieve.

[0032] (III) Bait preparation

[0033] Fresh oyster meat is ground using a small grinder (CG-8316, Long Plus), filtered through a 200-mesh screen, and the minced meat is used as feed for seedling cultivation.

[0034] (iv) Add Vitamin C

[0035] Seawater is introduced into the seedling ponds through a mesh filter. Each seedling pond is filled with seawater to a height of 0.5m, and 99% pure water-soluble vitamin C is added to the seawater at a dosage of 30mg / L to ensure it is fully dissolved.

[0036] (v) Seedling source and release

[0037] One-year-old diapause juvenile horseshoe crabs were provided by the Artificial Breeding Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. At this base, parent horseshoe crabs spawn in multiple batches each year from April to September. All fertilized eggs are incubated in indoor cement tanks, hatching into one-year-old juveniles in approximately 60 days. During the rearing process, the one-year-old juveniles experience drastic temperature fluctuations from winter to spring (sudden drops in temperature and large diurnal temperature variations). By late April of the following year, over 95% of the one-year-old juveniles remain in the one-year-old stage, i.e., diapause. The base can produce over 200,000 one-year-old diapause horseshoe crabs annually. Juveniles are randomly selected from the one-year-old diapause horseshoe crab population and reared according to the method of this invention. Their weight is 0.027 ± 0.002 g, and their carapace width is 0.580 ± 0.025 cm.

[0038] Four hundred 1-year-old horseshoe crabs were selected and transferred to an indoor rearing pond (8m×5m×1.5m) using a clean, specialized bionet for horseshoe crab seedling rearing. The transfer was carried out after 5:30 PM; the rearing density was 6.67 individuals / m². 3 (Seedling pond).

[0039] (vi) Management during the cultivation period

[0040] 1. Cultivation density management

[0041] Throughout the breeding process, the density of juvenile horseshoe crabs should be controlled to not exceed 8 individuals / m². 3 .

[0042] 2. Water quality management

[0043] During the cultivation period, the water temperature should be maintained at 28–31℃, salinity at 28–30, pH at 7.5–8.0, dissolved oxygen at 6.8–7.4 mg / L, ammonia nitrogen at less than 0.10 mg / L, and nitrite concentration at less than 0.005 mg / L.

[0044] 3. Feeding Management

[0045] Throughout the entire breeding process, oyster meat is used as feed. The oyster meat is ground and filtered into a paste, and fed once a day at 8:00 AM at a rate of 10% of the juvenile horseshoe crab's body weight.

[0046] 4. Water change and sludge removal

[0047] Throughout the entire rearing process, the water is changed every two days. First, use a sponge to wipe and clean the walls of the rearing pond to remove mucus and other dirt. At the same time, all the air stones and their hoses in the rearing pond need to be cleaned. Then, use a siphon with a filter membrane (to prevent backflow of juvenile horseshoe crabs) to suck up the dirt from the bottom of the entire pond. This operation replaces 90% of the original seawater. The fresh seawater described in step (II) is then used to prepare a rearing seawater containing 30 mg / L vitamin C and added to the rearing pond to the original volume to complete the water change operation.

[0048] 5. Other Management

[0049] The seedling ponds are placed in a breeding workshop with a covered shed, which has good ventilation and avoids direct sunlight. The cultivation light cycle maintains the natural light cycle.

[0050] Example 2

[0051] The difference between this embodiment and Embodiment 1 is that water-soluble vitamin C is added to the seawater at a dosage of 60 mg / L; after changing the water, it is necessary to add aquaculture seawater that has been prepared to contain 60 mg / L of vitamin C.

[0052] Example 3

[0053] Unlike Example 1, water-soluble vitamin C was added to the seawater at a concentration of 90 mg / L; after changing the water, it was necessary to add aquaculture seawater that had been prepared to contain 90 mg / L of vitamin C.

[0054] Comparative Example 1

[0055] Unlike Example 1, no vitamin C was added to the culture seawater.

[0056] During the cultivation of 1-year-old horseshoe crabs in Examples 1-3 and Comparative Example 1, molted shells 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; the results are shown in Table 1.

[0057] Survival rate SR (%) = 100 × (St - S0) / S0

[0058] Peeling rate (MR, %) = Mi / Cr × 100

[0059] In the formula: St and S0 are the number of surviving juvenile horseshoe crabs at the end and beginning, respectively; Mi is the cumulative number of molted individuals; and Cr is the total number of individuals in the group. The overall survival rate is the sum of the survival rates of 1-year-old diapause horseshoe crabs and 2-year-old molting juvenile horseshoe crabs.

[0060] Table 1

[0061] project Example 1 Example 2 Example 3 Comparative Example Overall survival rate (%) 38.67±6.66 49.17±1.04 68.33±2.52 33.50±4.77 Survival rate of 2-year-old juvenile horseshoe crabs (%) 69.54±5.93 89.32±2.08 91.68±1.90 57.63±0.86 Peeling rate (%) 24.33±3.69 31.17±3.40 24.00±1.00 20.50±7.09

[0062] like Figure 3 As shown, diapause-aged juvenile horseshoe crabs molted after being cultured in the aquaculture water with the addition of vitamin C. Table 1 shows that the culture method of the present invention can not only effectively improve the molting rate of diapause-aged juvenile horseshoe crabs but also improve their survival rate, that is, promote the development of diapause-aged Chinese horseshoe crabs, reduce their mortality rate, thereby obtaining high-quality 2-year-old horseshoe crab seedlings and improving aquaculture efficiency.

Claims

1. A breeding method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs, characterized in that, Includes the following steps: One-year-old horseshoe crabs in diapause were selected and cultured in seawater containing 30-90 mg / L of vitamin C. The term "diapause 1-year-old Chinese horseshoe crab" refers to an individual that hatched as a 1-year-old juvenile in September of the current year and has not molted into a 2-year-old horseshoe crab by April of the following year.

2. The cultivation method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs according to claim 1, characterized in that, The breeding density of Chinese horseshoe crabs is controlled at 5-8 individuals / m². 3 .

3. The cultivation method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs according to claim 2, characterized in that, During the breeding process, fresh oyster meat paste was fed to the juvenile horseshoe crabs once a day at a rate of 10% of their body weight.

4. The cultivation method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs according to claim 3, characterized in that, in During the cultivation process, the aquaculture water is changed every 2 days. The specific operation for changing the water is as follows: first, discard 90% of the volume of aquaculture water, and then add seawater containing 30~90mg / L of vitamin C back to the original volume.

5. The cultivation method for improving the molting rate and survival rate of diapause-aged Chinese horseshoe crabs according to claim 4, characterized in that, During the cultivation process, the temperature of the aquaculture water is maintained at 24~32℃.

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