A Navicula active substance for promoting the attachment and metamorphosis of bivalve mollusks and its application

Abstract

The present invention discloses a navicula active substance for promoting the attachment and metamorphosis of bivalve mollusks and its application. The navicula active substance is a polysaccharide of the active metabolite of navicula, which includes glucose, D-fucose, D-xylose, D-galactose, D-mannose, etc. The raw materials for its preparation are ecological and pollution-free. The prepared polysaccharide is non-toxic, and the preparation method is convenient and fast. It has been experimentally confirmed that the polysaccharide of the active metabolite of navicula can improve the attachment and metamorphosis rates of several late-veliger larvae such as scallops, oysters and ark clams, has the effect of inducing the attachment and metamorphosis of bivalve mollusks, has a high metamorphosis efficiency, can improve the economic benefits of bivalve mollusk farming, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of efficient ecological cultivation of aquatic fry, and particularly relates to a navicula active substance for promoting the attachment and metamorphosis of bivalve mollusks and its application. Background Art

[0002] Bivalve mollusks are important species in shallow sea aquaculture in China. In 2021, the aquaculture output reached 13.98 million tons, accounting for 92% of China's shellfish output, generating huge economic and social benefits. The acquisition of a large number of fry is crucial for large-scale aquaculture. Artificially cultivated fry can make up for the shortage of natural fry, and purposefully select and breed seeds to improve the efficiency of aquaculture enterprises. It is the main way to obtain fry in large-scale aquaculture. Bivalve mollusks are oviparous. From egg development to juvenile shellfish, they respectively go through the embryonic period, the planktonic larval period, and the attachment and metamorphosis period. Attachment and metamorphosis are important links in the development history of scallop larvae, and are sensitive periods for development and survival, during which their morphology and living habits change greatly. The larvae first secrete byssus to adhere and fix on a suitable attachment substrate, and then the velum degenerates, and gills and adductor muscles are developed. A new calcareous secondary shell is grown on the outer edge of the chitin shell (a sign of completed metamorphosis). During this process, the larvae change from a planktonic life to a life fixed by byssus, from filtering food with the velum to filtering food with gills, and from a chitin shell to a calcareous shell. In the attachment and metamorphosis stage, if the environmental factors are slightly unsuitable, the larvae will delay metamorphosis or die in large numbers, causing huge losses to shellfish seedling cultivation enterprises. Therefore, there is an urgent need to develop an efficient and ecological fry cultivation technology and method. Summary of the Invention

[0003] The purpose of the present invention is to provide a navicula active substance for promoting the attachment and metamorphosis of bivalve mollusks and its application. The present invention uses the active metabolite polysaccharide produced by navicula, which is extracted and purified and then used to promote the attachment and metamorphosis of bivalve mollusk larvae, thereby improving the economic benefits of bivalve mollusks.

[0004] To achieve the above invention purpose, the present invention adopts the following technical solutions to implement:

[0005] The present invention provides a navicula active substance for promoting the attachment and metamorphosis of bivalve mollusks, which is a navicula active metabolite polysaccharide, including glucose, D-fucose, D-xylose, D-galactose, and D-mannose.

[0006] Furthermore, the navicula active substance also includes gluconic acid, L-rhamnose, and glucosamine.

[0007] Furthermore, the preparation steps of the navicula active substance include:

[0008] (1) Centrifuge navicula in the exponential growth phase. After the obtained supernatant is filtered, anhydrous ethanol is added, and then centrifuged to remove the supernatant and obtain the precipitate;

[0009] (2) Remove the protein in the precipitate of step (1) using Sevag reagent, and centrifuge to obtain the supernatant.

[0010] (3) Dialyze the supernatant of step (2) using a dialysis bag, freeze the dialysate and then dry it to obtain the Navicula active substance.

[0011] Further, in step (1), the filtration is carried out using a 0.45 μm cellulose acetate membrane; the volume of the absolute ethanol is 2 - 5 times that of the supernatant.

[0012] Further, the conditions for protein removal in step (2) are: oscillation temperature: 4 - 6 °C, oscillation time: 15 min, number of repetitions: 3 times.

[0013] Further, in step (3), the dialysis bag is dialyzed in distilled water for 24 h, and the distilled water is changed every 6 hours; the freezing temperature is -80 °C.

[0014] The present invention also provides the application of the described Navicula active substance in promoting the attachment and metamorphosis of bivalve mollusks.

[0015] Further, the usage method of the Navicula active substance is: when the number of eyed larvae of bivalve mollusks reaches 60%, apply the Navicula active substance into the larval rearing pond.

[0016] Further, the usage concentration of the Navicula active substance is 2 - 5 g / L.

[0017] Further, the bivalve mollusks include scallops, oysters and ark clams.

[0018] Further, the Navicula active substance can cause calcium ion influx in oyster larvae.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] The present invention uses Navicula to prepare polysaccharides which are active metabolites of Navicula, including glucose, D-apiose, D-xylose, D-galactose, D-mannose, etc. Its raw materials are ecological and pollution-free, the prepared polysaccharides are non-toxic, and the preparation method is convenient and fast. It has been experimentally confirmed that in the environment of polysaccharides of Navicula active metabolites, there is obvious calcium ion influx in oyster larvae, and it has been confirmed that polysaccharides of Navicula active metabolites can improve the attachment and metamorphosis rate of late-stage larvae with shell tops of oysters, indicating that it has the effect of promoting the attachment and metamorphosis of bivalve mollusks, and the metamorphosis efficiency is high, which can improve the economic benefits of bivalve mollusk farming and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1It is the high performance liquid chromatography chart of the polysaccharide of the active metabolite of Navicula

[0022] Figure 2 It is about the Ca of oyster larvae in the polysaccharide of the active metabolite of Navicula and the control group 2+ Flow rate

[0023] Figure 3 It is the calcium ion flow rate of oyster larvae in the polysaccharide of the active metabolite of Navicula and the control group

[0024] Figure 4 It is the ethology of the larvae in the polysaccharide group of the active metabolite of Navicula

[0025] Figure 5 It is the ethology of the larvae in the control group

[0026] Figure 6 It is the oyster larvae in the experiment

[0027] Figure 7 It is the metamorphosis rate of oyster larvae in the polysaccharide of the active metabolite of Navicula and the control group Specific implementation mode

[0028] The technical solution of the present invention will be further described in detail in combination with the following specific examples

[0029] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies

[0030] Example 1: Extraction and analysis of the polysaccharide of the active metabolite of Navicula

[0031] The Navicula preserved in the laboratory was cultured on a large scale. The Navicula in the exponential growth phase was taken, centrifuged to obtain the supernatant, and the supernatant was filtered using a 0.45 μm cellulose acetate membrane. Then, anhydrous ethanol was used for alcohol precipitation according to 3 times the volume of the supernatant. After the obtained precipitate was centrifuged, the supernatant was removed. The volume was adjusted to 20 mL, 5 mL of Sevag reagent (the volume ratio of chloroform to n-butanol is 4:1) was added, shaken for 15 min, centrifuged at 5000 r / min for 10 min at 4 °C, and the upper layer clear liquid was collected; the Sevag treatment step was repeated 3 times. The extract was respectively filled into a pre-treated dialysis bag, dialyzed in distilled water for 24 h, and the distilled water was changed every 6 h. After dialysis was completed, it was transferred to a centrifuge tube, frozen in a -80 °C refrigerator, and freeze-dried with a freeze dryer to obtain the polysaccharide of the active metabolite of Navicula

[0032] The glycosyl composition of the polysaccharides, which are the active metabolites of Navicula, was analyzed. After the purified polysaccharides were completely hydrolyzed by acid and analyzed by HPLC, the proportion of monosaccharides in the EPS polysaccharides of the Navicula biofilm was obtained by comparing the peak areas of the monosaccharide standards.

[0033] The experimental results are as Figure 1 shown in Table 1. Among the glycosyl compositions of the polysaccharides, which are the active metabolites of Navicula, the largest proportion is glucose (43.03%), followed by D-fucose (24.19%), D-xylose (14.82%), D-galactose (12.61%), D-mannose (12.05%), while gluconic acid, L-rhamnose, and glucosamine account for relatively small proportions.

[0034] Table 1: Monosaccharide composition of the EPS polysaccharides of the Navicula biofilm

[0035]

[0036] Example 2

[0037] Non-invasive micro-test technology (NMT, Xuyue) was used to measure the Ca 2+ concentration gradient between two predetermined points using the corresponding microsensors. The Ca 2+ microsensors were pre-calibrated in Ca 2+ with concentrations of 0.5 mM and 0.05 mM. After the oyster eyed larvae were washed with the test solution (360 mM NaCl, 2.0 mM NaHCO3, 8.0 mM KCl, 0.1 mM Na2SO4, 0.5 mM CaCl2, pH 8.1) for 20 min to adapt them, the polysaccharides, which are the active metabolites of Navicula extracted in Example 1, were added. The Ca 2+ flux microsensor was placed near the shell edge (about 3 µm) to detect the calcium ion exchange of the oyster larvae. The Ca 2+ flux data was exported from the imFluxes V2.0 software.

[0038] Using the oyster eyed larvae in the test solution as the control group and the test solution added with the polysaccharides, which are the active metabolites of Navicula as the treatment group, the calcium ion flux in the larvae was monitored. The results are as Figure 2 shown in Figure 3 and indicate that there is an obvious calcium ion influx in the oyster larvae in the environment of the polysaccharides, which are the active metabolites of Navicula, and the difference is significant.

[0039] Example 3

[0040] 1. Behavioral statistics were performed on the oyster larvae. The results are as Figure 4 shown in Figure 5As shown in the figure, the behavioral data of oyster larvae in the face of different groups of attachment environments show that when the larvae are placed in the attachment environment for 24 hours, 30% of the larvae are in the swimming state in the polysaccharide group of the active metabolites of Navicula sp., 64.58% of the larvae are at the bottom of the polysaccharide group, and 5.42% of the larvae are in the crawling state, exploring suitable attachment sites. In the control group, 67.59% of the larvae are in the swimming state, and only 32.41% of the larvae are located at the bottom of the attachment substrate. At 48 hours, 3.33% of the larvae in the polysaccharide group completed metamorphosis into juveniles, the number of larvae at the bottom increased to 68.33%, and the number of swimming larvae began to decrease. In the control group, only 1.52% of the larvae completed metamorphosis, and the number of swimming larvae (45.00%) was more than that in the polysaccharide group (23.33%), and the proportion of larvae at the bottom (50.91%) was less than that in the active substance group (68.335%). At 72 h, 120 h and 168 h, the results generally showed that the number of juveniles in the polysaccharide group continued to increase (15.00%, 30.56%, 51.67%) and was higher than that in the control group (5.00%, 14.70%, 29.72%), and the number of swimming larvae continued to decrease (21.67%, 5.00%, 3.33%) and was less than that in the control group (23.33%, 12.88%, 10.19%). At 216 h, 69.63% of the larvae in the polysaccharide group completed metamorphosis into juveniles, while only 43.33% in the control group ( P <0.01). It shows that the polysaccharide of the active metabolites of Navicula sp. can accelerate the metamorphosis process of oyster larvae.

[0041] 2. Cultivate oysters. When the number of eyed larvae of oysters reaches 60%, release the polysaccharide of the active metabolites of Navicula sp. extracted in Example 1 into the larval cultivation pond at a concentration of 2-5 g / L for conventional cultivation, and detect the metamorphosis rate of the eyed larvae of Crassostrea gigas.

[0042] The experimental results of the polysaccharide of the active metabolites of Navicula sp. on the metamorphosis of oysters are as Figure 6 and Figure 7 shown. The metamorphosis rate of the control group is 5.3%, and that of the polysaccharide group of the active metabolites of Navicula sp. is 16.9%. It shows that the polysaccharide of the active metabolites of Navicula sp. can significantly improve the attachment and metamorphosis rate of the late-stage larvae of oyster shell tops.

[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A Navicula active substance for promoting the attachment and metamorphosis of bivalve mollusks, characterized in that, The Navicula active substance is a polysaccharide of the active metabolite of Navicula, including glucose, D-fucose, D-xylose, D-galactose, and D-mannose; the Navicula active substance also includes gluconic acid, L-rhamnose, and glucosamine; the Navicula active substance can cause calcium ion influx in oyster larvae; The bivalve is an oyster; The preparation steps of the Navicula active substance include: (1) Centrifuge Navicula in the exponential growth phase. After filtering the obtained supernatant, add absolute ethanol, centrifuge to remove the supernatant, and take the precipitate; The filtration uses a 0.45 μm cellulose acetate membrane; (2) Use Sevag reagent to remove the protein in the precipitate of step (1), and centrifuge to take the supernatant; (3) Dialyze the supernatant of step (2) using a dialysis bag, freeze the dialysate and dry it to obtain the Navicula active substance.

2. The navicula active substance according to claim 1, characterized in that, In step (1), the volume of the absolute ethanol is 2-5 times that of the supernatant.

3. The navicula active substance according to claim 1, characterized in that, The conditions for protein removal in step (2) are: oscillation temperature: 4-6 °C, oscillation time: 15 min, number of repetitions: 3 times.

4. The navicula active substance according to claim 1, characterized in that, In step (3), the dialysis bag is dialyzed in distilled water for 24 h, and the distilled water is changed every 6 hours; the freezing temperature is -80 °C.

5. Use of the Navicula active substance according to any one of claims 1-4 in promoting the attachment and metamorphosis of bivalve mollusks, characterized in that, The bivalve is an oyster; 6. The application according to claim 5, wherein The usage method of the Navicula active substance is: when the number of eyed larvae of bivalves reaches 50%-60%, apply the Navicula active substance to the larval culture pond; 7. The application according to claim 6, wherein The usage concentration of the Navicula active substance is 2-5 g / L.

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