Use of navicula active substance for accelerating attachment metamorphosis of bivalve

Abstract

To provide an active substance having a function of enhancing an attachment metamorphosis rate of umbo later-stage larva of scallop, oyster, ark shell, etc., and inducing attachment metamorphosis, and a use method thereof.SOLUTION: An attachment metamorphosis enhancing method of bivalve which includes scallop, oyster, and ark shell includes adding glucose, D-fucose, D-xylose, D-galactose, D-mannose, gluconic acid, L-rhamnose, and glucosamine which are navicula active substance metabolite polysaccharides to a breeding water body in a culture tank for eyespot larva of the bivalve.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention belongs to the field of efficient ecological cultivation techniques for aquatic seedlings, and specifically relates to the use of active substances of Grateloupia filicina to promote the attachment and metamorphosis of bivalves.

Background Art

[0002] Bivalves are an important type of shallow-sea aquaculture in our country. The aquaculture production volume in 2021 reached 13.98 million tons, accounting for 92% of the shellfish production volume in our country, generating huge economic and social benefits. Among them, the acquisition of a large number of seedlings has made great contributions to large-scale aquaculture. Artificially cultivated seedlings can make up for the shortage of natural seedlings, and intentionally selecting and breeding to increase the profits of aquaculture enterprises is the main method for obtaining seedlings in large-scale aquaculture. Bivalves are oviparous and go through the embryonic stage, planktonic larval stage, and attachment and metamorphosis stage from egg development to juvenile clams. Attachment and metamorphosis are important links in the larval development history of scallops, a sensitive period for development and survival, during which their morphology and living habits change greatly. The larvae first secrete byssus to adhere and fix to a suitable attachment substrate, and then the veliger degenerates, the gills and adductor muscles develop, and a new secondary shell of calcium carbonate grows on the outer edge of the chitin shell (signs of complete metamorphosis). During this process, the larvae transition from a planktonic life to a sessile life using byssus, from filtering food with the veliger to filtering food with the gills, and from a chitinous shell to a calcium carbonate shell. At the stage of attachment and metamorphosis, if environmental factors are slightly abnormal, the larvae may delay metamorphosis or die in large numbers, causing great losses to shellfish seedling breeding enterprises. Therefore, it is urgent to develop efficient and ecological seedling breeding techniques and methods.

Summary of the Invention

Problems to be Solved by the Invention

[0003] An object of the present invention is to provide an active substance of Grateloupia filicina that promotes the attachment and metamorphosis of bivalves and its use. The present invention utilizes the active metabolite polysaccharide produced by Grateloupia filicina, extracts and purifies it, and then uses it to promote the attachment and metamorphosis of bivalve larvae, and further increases the economic benefits of bivalves.

Means for Solving the Problem

[0004] In order to achieve the above object of the invention, the present invention is realized by using the following technical solutions.

[0005] The present invention provides a Laminaria japonica active substance that promotes the attachment and metamorphosis of bivalves, which is a Laminaria japonica active metabolite polysaccharide and contains glucose, D-fucose, D-xylose, D-galactose, and D-mannose.

[0006] Furthermore, gluconic acid, L-rhamnose, and glucosamine are further contained in the Laminaria japonica active substance.

[0007] Furthermore, the preparation steps of the Laminaria japonica active substance are as follows: (1) Centrifuge Laminaria japonica in the exponential growth phase, filter the obtained supernatant, add anhydrous ethanol, centrifuge, discard the supernatant, and collect the precipitate; (2) Remove the protein in the precipitate of step (1) with Sevag reagent, and centrifuge to collect the supernatant; (3) Dialyze the supernatant of step (2) using a dialysis bag, freeze the dialysate, and then dry it to obtain the Laminaria japonica active substance.

[0008] Furthermore, a 0.45 μm cellulose acetate film is used for filtration in step (1), and the volume of the anhydrous ethanol is 2 - 5 times that of the supernatant.

[0009] Furthermore, as the conditions for removing the protein in step (2), the shaking temperature is 4 - 6 °C, the shaking time is 15 min, and the number of repetitions is 3 times.

[0010] Furthermore, the dialysis bag in step (3) is dialyzed in distilled water for 24 h, and the distilled water is changed every 6 h. The freezing temperature is -80 °C.

[0011] The present invention further provides the use of the above-mentioned Grifola frondosa active substance for promoting the attachment and metamorphosis of bivalves.

[0012] Furthermore, the method for using the above-mentioned Grifola frondosa active substance is that when the number of eyed larvae of bivalves reaches 60%, the Grifola frondosa active substance may be administered into the larval culture tank.

[0013] Furthermore, the use concentration of the above-mentioned Grifola frondosa active substance is 2 - 5 g / L.

[0014] Furthermore, the above-mentioned bivalves include scallops, oysters and ark clams.

[0015] Furthermore, the above-mentioned Grifola frondosa active substance may cause the influx of calcium ions in the larvae of oysters.

Advantages of the Invention

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0017] The present invention prepares Grifola frondosa active metabolite polysaccharides containing glucose, D-fucose, D-xylose, D-galactose, D-mannose, etc. by using Grifola frondosa. Its raw materials are ecological, pollution-free, the prepared polysaccharides are non-toxic, and the preparation method is convenient and rapid. Through experiments, it is confirmed that there is an obvious influx of calcium ions in the larvae of oysters under the environment of Grifola frondosa active metabolite polysaccharides. Also, it is confirmed that Grifola frondosa active metabolite polysaccharides can increase the attachment and metamorphosis rate of late-stage larvae of oyster shell tops, have the effect of promoting the attachment and metamorphosis of bivalves, and have a high metamorphosis efficiency, which can increase the economic benefits of bivalve aquaculture and shows good prospects for use.

Brief Description of the Drawings

[0018]

Figure 1

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Mode for Carrying Out the Invention

[0019] The technical solution of the present invention will be described in more detail with reference to the following specific examples.

[0020] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0021] Example 1: Extraction and Analysis of the Polysaccharide of the Active Metabolite of Funagata keiso

[0022] The hornwort cultured and preserved in the laboratory was expanded and cultured, and the culture solution containing hornwort in the exponential growth phase was taken, centrifuged to obtain the supernatant, the supernatant was filtered through a 0.45 μm cellulose acetate film, and alcohol precipitation was performed using anhydrous ethanol at three times the volume of the supernatant. After centrifuging the obtained precipitate, the supernatant was discarded. 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, the supernatant was collected, and the Sevag treatment step was repeated three times. The extracts were respectively put into pre-treated dialysis bags, dialyzed in distilled water for 24 h, and the distilled water was changed every 6 h. After dialysis, they were transferred to centrifuge tubes, frozen in a refrigerator at -80 °C, and freeze-dried with a freeze dryer after freezing to obtain the active metabolite polysaccharide of hornwort.

[0023] Glycosyl group composition analysis was performed on the active metabolite polysaccharide of hornwort. After complete acid hydrolysis and HPLC analysis of the purified polysaccharide, the ratio of the monosaccharide composition in the EPS polysaccharide of the hornwort biofilm was obtained by comparing with the peak area of the monosaccharide standard product.

[0024] The experimental results are shown in Figure 1 and Table 1. Among the glycosyl group compositions of the active metabolite polysaccharide of hornwort, the proportion of glucose is the highest (43.03%), followed by D-fucose (24.19%), D-xylose (14.82%), D-galactose (12.61%), D-mannose (12.05%) in this order, and gluconic acid, L-rhamnose and glucosamine account for relatively small proportions.

[0025]

Table 1

[0026] Example 2 Non-destructive microtechnology (NMT, Xuyue) uses the corresponding microsensor to measure the Ca 2+ concentration gradient between two predetermined points. Ca 2+The microsensor is calibrated in advance with 0.5 mM and 0.05 mM Ca 2+ The test solution (360 mM NaCl, 2.0 mM NaHCO 3 , 8.0 mM KCl, 0.1 mM Na 2 SO 4 , 0.5 mM CaCl 2 , pH 8.1) was used to wash the eyed larvae of oysters and allow them to adapt for 20 min. Then, the polysaccharide of the active metabolite of Ganoderma tsugae extracted in Example 1 was added. The Ca 2+ flux microsensor was placed near the shell edge (about 3 μm) to detect the exchange status of calcium ions in the oyster larvae. The Ca 2+ flux data was derived from im Fluxes V2.0 software.

[0027] Using the eyed larvae of oysters in the test solution as the control group and the group with the addition of the polysaccharide of the active metabolite of Ganoderma tsugae to the test solution as the treatment group, the calcium ion flux in the larvae was monitored. The results are shown in Figures 2 and 3. Under the environment of the polysaccharide of the active metabolite of Ganoderma tsugae, there was an obvious influx of calcium ions in the oyster larvae, and the difference was significant.

[0028] Example 3 1. Ethological statistics were conducted on oyster larvae. The results are shown in Figures 4 and 5. From the ethological data of different groups of oyster eyed larvae in different attachment environments, when the oyster eyed larvae were placed in the attachment environment for 24 hours, in the active metabolite polysaccharide group of Sargassum fusiforme (also called the polysaccharide group), 30% of the larvae were in a swimming state, 64.58% of the larvae were at the bottom of the polysaccharide group, and 5.42% of the larvae were in a crawling state, searching for a suitable attachment place. Among the control group, 67.59% of the larvae were in a swimming state, and only 32.41% of the larvae were at the bottom of the attachment substrate. After 48 hours, in the polysaccharide group, 3.33% of the larvae completed metamorphosis and became juvenile oysters, the number of larvae at the bottom increased to 68.33%, and the number of swimming larvae began to decrease. Among the control group, only 1.52% of the larvae metamorphosed, the number of swimming larvae (45.00%) was more than that of the polysaccharide group (23.33%), and the proportion of larvae at the bottom (50.91%) was less than that of the polysaccharide group (68.335%). From the results at 72 hours, 120 hours, and 168 hours, overall, the number of juvenile oysters in the polysaccharide group continued to increase (15.00%, 30.56%, 51.67%) and was higher than that of 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 smaller than that of the control group (23.33%, 12.88%, 10.19%). After 216 hours, 69.63% of the larvae in the polysaccharide group completed metamorphosis and became juvenile oysters, while in the control group, it was only 43.33% (P < 0.01). It was shown that the active metabolite polysaccharide of Sargassum fusiforme can accelerate the metamorphosis process of oyster larvae.

[0029] 2. Oysters were cultured. When the number of oyster eyed larvae reached 60%, the active metabolite polysaccharide of Sargassum fusiforme extracted in Example 1 was placed in the larval culture tank at a concentration of 2 - 5 g / L for normal aquaculture, and the metamorphosis rate of oyster eyed larvae was detected.

[0030] The experimental results of the active metabolite polysaccharide of Sargassum fusiforme on oyster metamorphosis are shown in Figures 6 and 7. The metamorphosis rate of the control group was 5.3%, and that of the active metabolite polysaccharide group of Sargassum fusiforme was 16.9%. This showed that the active metabolite polysaccharide of Sargassum fusiforme can significantly improve the attachment and metamorphosis rate of late-stage larvae with shell tops of oysters.

[0031] The above embodiments are only used for explaining the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the foregoing embodiments or equivalently replace some of their technical features, and these modifications or replacements do not deviate from the spirit and scope of the technical solutions claimed by the present invention for the corresponding technical solutions.

Claims

1. A use of an Ardisia crenata active substance for promoting attachment metamorphosis of bivalve mollusks, characterized in that when the number of bivalve eyespot larvae reaches 50%-60%, the Ardisia crenata active substance is administered to a culture water body in a bivalve eyespot larvae culture tank, the Ardisia crenata active substance is used at a concentration of 2-5 g / L, the Ardisia crenata active substance is an Ardisia crenata active metabolite polysaccharide, the Ardisia crenata active metabolite polysaccharide contains glucose, D-fucose, D-xylose, D-galactose, D-mannose, gluconic acid, L-rhamnose and glucosamine, and the bivalves include scallops, oysters and ark shells.

2. The preparation step of the active substance of Funagatakeisou is (1) centrifuging exponentially growing F. fasciatus, filtering the resulting supernatant, adding anhydrous ethanol, centrifuging, discarding the supernatant, and collecting the precipitate; Step (2) of removing the proteins in the precipitate of step (1) with Sevag reagent and centrifuging to obtain the supernatant; The use according to claim 1, characterized in that it also includes a step (3) of dialysing the supernatant of step (2) using a dialysis bag, freezing the dialysate and then drying it to obtain Fuchidoide active substances.

3. The active substance of Funagatakeisou described in claim 2, characterized in that a 0.45 μm cellulose acetate film is used for filtration in step (1), and the volume of the anhydrous ethanol is 2-5 times that of the supernatant.

4. The Fungal spore active substance according to claim 2, characterized in that in step (2), the conditions for removing protein are a shaking temperature of 4-6°C, a shaking time of 15 minutes, and a number of repetitions of 3 times.

5. The active substance of Funagatake Dioscorea as described in claim 2, characterized in that in step (3), the dialysis bag is dialyzed in distilled water for 24 hours, the distilled water is replaced every 6 hours, and the freezing temperature is -80°C.

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