Chaetoceros muelleri and its application as feed for Penaeus vannamei

Abstract

The present invention provides a Chaetoceros muelleri and its application as a feed for Penaeus vannamei, that is, a Chaetoceros muelleri with high temperature resistance and its application, which can be used as a biological bait for Penaeus vannamei in high temperature seasons. The provided Chaetoceros muelleri has a deposit number of CCTCC NO: M2024686. The microalgae screened by the present invention can withstand high temperatures and have a high protein content. It performs outstandingly in a feeding intensity test of Penaeus vannamei seedlings, can be used as an alternative bait for aquaculture, and provides a new biological bait for feeding aquatic animals in summer.

Description

Technical Field

[0001] The invention belongs to the technical field of microalgae screening and application, and particularly relates to Chaetoceros muelleri and application of the same as white shrimp feed. Background Art

[0002] The whiteleg shrimp is a high-yield shrimp species that has attracted much attention in the aquaculture industry and has become one of the three major shrimp aquaculture varieties in the world. It is mainly distributed along the Pacific coast of America and is the main aquaculture target in American countries such as Ecuador. It is also widely aquacultured in the Yellow Sea and Bohai Sea in China. In 2023, China's whiteleg shrimp aquaculture area was 165,651 thousand hectares. Whiteleg shrimp is not only of high economic value and rich in nutrition, but also deeply loved by consumers because of its thin shell, fat body, warm nature and sweet taste, and the effect of nourishing the kidney and strengthening yang. Stable market demand has driven the aquaculture output of whiteleg shrimp in China. In recent years, it has continued to remain above 1 million tons, and the aquaculture output in 2023 reached 2.0987 million tons.

[0003] Algae are essential in the cultivation of white shrimp. Algae in the water can inhibit the number and reproduction of harmful bacteria in the water, provide sufficient oxygen for shrimp, effectively balance the pH, create a good water color and appropriate transparency, inhibit the reproduction of harmful algae at the bottom of the pond, provide nutritious bait for shrimp fry, improve the survival rate of shrimp, and provide a healthy ecological environment for white shrimp. Temperature is one of the most important ecological factors affecting the growth and reproduction of marine microalgae. The greater the frequency and amplitude of temperature fluctuations, the more unfavorable it is to the growth rate of bait algae and the increase of algae cell density.

[0004] Under high temperature conditions in summer, the water temperature of aquaculture ponds in Zhejiang Province can reach 34°C or above, and the water temperature of artificial bait cultivation ponds can sometimes exceed 40°C. High temperatures affect the reproduction and growth of bait algae, causing the death of bait microalgae. This makes it difficult to meet the demand for live bait for aquatic products during summer seedling cultivation, and on the other hand, it will also deteriorate the aquaculture water quality, leading to an increased risk of disease outbreaks. Summary of the invention

[0005] The invention aims to provide a Chaetoceros muelleri and its application as a feed for Penaeus vannamei, that is, a Chaetoceros muelleri with high temperature resistance and its application, which can be used as a biological bait for Penaeus vannamei in high temperature seasons.

[0006] The Chaetoceros spp. provided by the present invention is Chaetoceros spp. 172 strains, which was deposited in the China Type Culture Collection Center of Wuhan University, Wuhan, China on April 12, 2024, with a deposit number of CCTCC NO: M2024686.

[0007] The Chaetoceros spp. 172 strains provided by the present invention have a high temperature resistance effect. As an example, the high temperature resistance is 35-40°C.

[0008] In another aspect, the present invention provides a use of the Chaetoceros spp. 172 strain as a bait for aquatic animals;

[0009] The aquatic animal, as specifically described in the embodiment, is the whiteleg shrimp.

[0010] In yet another aspect, the present invention provides a method for cultivating Penaeus vannamei, which uses the selected Chaetoceros muelleri strain 172 as bait to feed the Penaeus vannamei.

[0011] The microalgae screened by the present invention can withstand high temperatures and have a high protein content, and performs outstandingly in a test of feeding intensity of white shrimp seedlings, and can be used as an alternative bait for aquaculture, providing a systematic biological bait for feeding aquatic animals in summer. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Morphological images of Chaetoceros muelleri under optical microscopy, scanning electron microscopy and cryo-electron microscopy, where A is optical microscopy, B is scanning electron microscopy, and C is cryo-electron microscopy;

[0013] Figure 2 : Growth curves of 172 strains under different optimized conditions (38℃);

[0014] Figure 3 : Growth rate diagram of 172 strains under different optimized conditions (45℃). DETAILED DESCRIPTION

[0015] Chaetoceros muelleri is a marine diatom belonging to the order Bacillariophyceales of the phylum Bacillariophyceae. It is a small marine floating diatom with thin cell walls and golden yellow algae. It usually lives alone or in groups of 2-3 cells. Because Chaetoceros muelleri contains a large amount of nutrients such as fatty acids and proteins, it is widely used in the hatching of larvae of fish, shellfish and shrimp.

[0016] The genus Chaetoceros can grow and reproduce normally at 25-34℃, its suitable growth temperature is 28-31℃, and the optimal growth temperature is 31℃. The research results show that the optimal temperature for Chaetoceros biomass production is 30℃, but it can maintain at least 66% of the maximum yield at 20-35℃. When the temperature is higher than 35℃, the growth rate of Chaetoceros slows down and then dies quickly.

[0017] The present invention collects water samples from a Chaetoceros muckerii cultivation pool of Ningbo Xiangshan Harbor Aquatic Seed Co., Ltd. The water temperature when the algae are collected is 43°C, and most of the algae cultivated in the water have decayed. Microalgae single cells are separated by micromanipulation, and Chaetoceros muckerii strains are obtained by single cell culture. The high temperature resistance performance and protein content of the algae are tested and the target high temperature resistant algae strain is finally screened out.

[0018] The present invention is described in detail below in conjunction with embodiments and drawings.

[0019] Example 1: Screening of high temperature resistant algae strains

[0020] 1. Isolation and cultivation of microalgae

[0021] Water samples were retrieved from the bait culture pond of Xiangshan Harbor Aquatic Seed Co., Ltd. in summer. The real-time water temperature was 43°C. The cultivated Chaetoceros muelleri had been seriously decayed after long-term high temperature. Immediately after retrieval, the living algae cells that survived the high temperature stress were separated by microscopy. The algae were cultured in f / 2 medium (Guillard et al, 1962), with a temperature of 25°C, a salinity of 32, a light-dark cycle of 12h / 12h, and a light intensity of 12μmol / (cm 2 ·s). Shake the algae solution once a day to prevent the growth of cells adhering to the wall from interfering with the experimental results.

[0022] Four strains of Chaetoceros muelleri were isolated by micromanipulation technology. They were named 170, 171, 172, and 173. After 48 hours of single-cell culture, the color of each strain was observed to be green, the color was pure, the cell size was consistent under microscopic examination, and there was no protozoan contamination.

[0023] 2. High temperature resistance test of algae strains

[0024] In this experiment, two temperature groups, A (38°C) and B (45°C), were set to test the high temperature resistance of the isolated algae strains. Three parallels were set up for each group and each algae strain. 100mL of algae liquid was taken from the algae strains that reached the logarithmic growth period after isolation and culture to a 250mL triangular flask. Groups A and B were placed in a constant temperature and light incubator at 38°C and 45°C, respectively, with a light-dark cycle of 24h / 0h, and the effective light intensity of groups A and B was 16μmol / (cm 2 ·s), and the absorbance at 681 nm was used to characterize the number of cells.

[0025] The isolated algae strains were cultured in 38℃ water, and it was found that all the isolated algae strains could survive at 38℃. From the beginning of inoculation to the 7th day, the number of cells of each algae strain increased; compared with the 7th day, the number of cells of strains 172 and 173 continued to increase on the 9th day, while the number of cells of strains 170 and 171 did not change much; according to the trend of the growth curve, the number of cells of strains 172 and 173 may continue to grow. During the culture period, the number of cells of strains 172 and 173 increased almost linearly, and the growth state was optimal. It is speculated that these two algae strains have a good adaptability to high temperature (38℃) (Table 1).

[0026] Table 1: Survival and growth performance of Chaetoceros muelleri under the culture temperature of 38°C

[0027]

[0028] When the two dominant algae strains isolated at 38°C were cultured at 45°C, the growth state of the algae strains was completely different from that at 38°C. As shown in Table 2, after 9 days of culture at 45°C, a large number of algae strains died in 173 strains, while 172 strains began to grow slowly after 5 days of decline, proving that 172 strains can adapt to 45°C.

[0029] Table 2 Survival and growth performance of Chaetoceros muelleri under culture temperature of 45°C

[0030]

[0031] Example 2: Determination of protein content of algae strains

[0032] The protein content of the selected thermostable bait microalgae was determined by Kjeldahl method.

[0033] The average protein content of Chaetoceros muelleri was 26.71%. The protein contents of the four isolated Chaetoceros muelleri strains were significantly different (P<0.05), with 171 having the lowest protein content and 172 having the highest protein content, as shown in Table 3.

[0034] Table 3: Protein content of thermotolerant Chaetoceros muelleri

[0035]

[0036]

[0037] Example 3: Optimization of culture conditions for high temperature resistant algae strains

[0038] The present invention optimizes the culture medium for strain 172, and the optimization indicators are pH value and nitrogen-phosphorus ratio (NaNO3: Na2HPO4). Three pH gradients are set: pH=7, pH=8, pH=9, and three N / P ratio gradients are set: 12:1, 13:1, 14:1. Cross-experiments are conducted on pH value and N / P, with a total of 9 gradients. 172 is cultured in a constant temperature shaking incubator at 38°C, with a light-dark cycle of 24h / 0h and a light intensity of 12μmol / (cm 2 ·s).

[0039] from Figure 2 and Figure 3 It can be seen that when the pH value range is 7-9 and the nitrogen-phosphorus ratio is 12:1, 13:1, and 14:1, the cell number and growth rate of 172 both show an increasing trend. This may be because the pH value and the nitrogen-phosphorus ratio are within the tolerance range of Chaetoceros muelleri. Chaetoceros muelleri can grow under conditions of pH 3-9. Studies have found that the optimal pH range for the growth of golden algae is 7.0-9.0. When it deviates from this range, the growth of the algae and the metabolic activities in the body will be inhibited. Chaetoceros muelleri grows fastest when the nitrogen-phosphorus ratio is equal to 16:1.

[0040] Normally, the pH in seawater is relatively stable (pH ≈ 8.2), but due to natural factors, the pH value of seawater will fluctuate. The pH value will affect lipid productivity and have a wide range of effects on cell metabolism. Nitrogen and phosphorus nutrients are important factors in the growth of algae. Phosphorus is essential for biomass production. Therefore, this experiment optimized the nitrogen-phosphorus ratio and pH value in the culture medium. The results showed that when N / P = 14:1 and pH = 7, the growth rate of algal cells was the fastest and the number of cells was the highest. Therefore, the most suitable culture medium for 172 strains at 38°C was N / P = 14:1 and pH = 7.

[0041] Example 4: Experiment on the cultivation of 172 algae strains in summer

[0042] 400 10-day-old whiteleg shrimp seedlings with a body length of about 1.0 cm were selected, and 300 healthy seedlings with good growth status were selected and randomly divided into 5 groups, with 60 whiteleg shrimp seedlings in one group and 3 parallel replicates in each group.

[0043] The shrimps were placed in 50L culture containers, with 15cm clean sea sand on the bottom. The filtered seawater was used to change the water once a day at 6:00am and aerated for 7-10 days. The fresh culture medium of strains 170, 171, 172, and 173 was used to feed the shrimps at 5:30pm every day. The initial concentration of the microalgae in the bait was 2×10 6 -3.5×10 6CFU / mL. The blank group served as the control group. The feeding experiment lasted for 5 days, and sampling began on the third day after the experiment began. Seawater samples were taken three times at different locations in the culture pond 2 hours after feeding to test the turbidity of seawater.

[0044] Among the high-temperature-resistant Chaetoceros muelleri strains screened out, there was a significant difference (P < 0.05) in the feeding intensity of 10-day-old whiteleg shrimp seedlings (about 1.0 cm in body length) on the four high-temperature-resistant Chaetoceros muelleri strains screened out. As can be seen from Table 4, the feeding intensity of 10-day-old whiteleg shrimp seedlings on 172 strains was the highest.

[0045] Table 4: Average feeding intensity of Penaeus vannamei on the thermotolerant Chaetoceros muelleri

[0046]

[0047] After one month of experiment, 10 healthy and active shrimp fry were collected from each group, and the weight, body length and other body size data of the shrimp fry were measured after the surface water was sucked dry. As shown in Table 5, the body size of the white shrimp in the different algae strain feeding groups was significantly different (P < 0.05). The weight and body length of the white shrimp fed with 172 algae strains were significantly higher than those of other groups, which showed a growth advantage.

[0048] Table 5: Statistics of growth of Penaeus vannamei in each algae strain group

[0049]

[0050] In summary, the heat-resistant Chaetoceros muelleri isolated by the present invention has a high protein content. Through the feeding experiment of white shrimp, it is confirmed that the white shrimp seedlings have a high feeding intensity on it, and it has a promoting effect on the growth of shrimp seedlings. Therefore, it can be used as a bait microalgae for heat-resistant white shrimp seedlings.

Claims

1. A species of Chaetoceros muelleri ( Chaetoceros spp.), characterized in that The Chaetoceros muehii ( Chaetoceros spp.) is deposited with CCTCC NO:M2024686.

2. The Chaetoceros muehii of claim 1 ( Chaetoceros spp.) as bait for aquatic animals.

3. The use according to claim 2, characterized in that The aquatic animal is Penaeus vannamei.

4. A method for cultivating Penaeus vannamei, characterized in that: The method is to use the Chaetoceros muelleri described in claim 1 ( Chaetoceros spp.) as bait to feed whiteleg shrimp.

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