Use of *Cylindrica pulcherrima* active substances to promote bivalve attachment and metamorphosis.

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 the active substances of Porphyra yezoensis 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 scale aquaculture. Artificially cultivated seedlings can supplement the shortage of natural seedlings. Intentionally selecting and breeding to increase the profits of aquaculture enterprises is the main method for obtaining seedlings in scale aquaculture. Bivalves are oviparous and go through the embryonic stage, floating larval stage, and attachment and metamorphosis stage from egg development to juvenile bivalves. Attachment and metamorphosis are an important part of the development history of scallop larvae, a sensitive period for development and survival, and 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 calcium carbonate secondary shell grows on the outer edge of the chitin shell (signs of completed metamorphosis). In this process, the larvae transition from a floating life to a sessile life using byssus, from filtering food with the veliger to filtering food with the gills, and from a chitin 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 cultivation enterprises. Therefore, it is urgent to develop efficient and ecological seedling cultivation technologies 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 Porphyra yezoensis that promotes the attachment and metamorphosis of bivalves and its use. The present invention utilizes the active metabolite polysaccharide produced by Porphyra yezoensis, 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] To achieve the above-mentioned objectives of the invention, the present invention is realized using the following technical solutions.

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

[0006] Furthermore, the aforementioned diatomaceous earth active substance also contains gluconic acid, L-rhamnose, and glucosamine.

[0007] Furthermore, the preparation step of the Funagata diatom active substance is (1) The exponential growth stage of the diatomaceous rhizome is centrifuged, the resulting supernatant is filtered, anhydrous ethanol is added, the supernatant is discarded and the precipitate is removed, (2) Remove the protein from the precipitate in step (1) using Sevag reagent, and take the supernatant by centrifugation. (3) Dialysis of the supernatant from step (2) using a dialysis bag, freezing the dialysate and then drying it to obtain the Funagata diatom active substance.

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

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

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

[0011] The present invention further provides the use of the aforementioned diatomaceous rhizome active substance for promoting bivalve attachment and metamorphosis.

[0012] Furthermore, the method for using the diatomaceous rhizome active substance is to administer the diatomaceous rhizome active substance into the larval culture tank when the number of eyespot larvae of the bivalve reaches 60%.

[0013] Furthermore, the concentration of the diatomaceous earth active substance used is 2-5 g / L.

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

[0015] Furthermore, the aforementioned diatom activity may induce an influx of calcium ions into oyster larvae. [Effects of the Invention]

[0016] Compared to the conventional technology, the present invention has the following advantages and beneficial effects.

[0017] This invention provides a method for preparing polysaccharides of active metabolites from the diatomaceous fossil, containing glucose, D-fucose, D-xylose, D-galactose, D-mannose, etc., using the diatomaceous fossil. The raw materials are ecological and free from contamination, the prepared polysaccharides are non-toxic, and the preparation method is convenient and rapid. Experiments have confirmed that oyster larvae undergo a clear influx of calcium ions in the environment of the polysaccharides of active metabolites from the diatomaceous fossil. Furthermore, it has been confirmed that the polysaccharides of active metabolites from the diatomaceous fossil are effective in promoting bivalve detachment and metamorphosis, and that they have a high metamorphosis efficiency, can increase the economic benefits of bivalve farming, and show promising prospects for use. [Brief explanation of the drawing]

[0018] [Figure 1] This is a high-performance liquid chromatogram of polysaccharide active metabolites from *Diospyros tinctoria*. [Figure 2]The Ca2+ flow rate in the polysaccharide group of the active metabolite of Funagatakeiso in oyster larvae and the control group. [Figure 3] The calcium ion flow rate in the polysaccharide group of the active metabolite of Funagatakeiso in oyster larvae and the control group. [Figure 4] The larval behavioral science of the polysaccharide group of the active metabolite of Funagatakeiso. [Figure 5] The larval behavioral science of the control group. [Figure 6] Oyster larvae during the experiment. [Figure 7] The metamorphosis rate of oyster larvae in the polysaccharide group of the active metabolite of Funagatakeiso and the control group.

Modes for Carrying Out the Invention

[0019] The technical solution of the present invention will be further described in 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 Funagatakeiso

[0022] Diospyrostrata var. japonica, which had been preserved in the laboratory, was cultured on a large scale. The culture medium containing Diospyrostrata var. japonica in the exponential growth stage was taken, centrifuged, and the supernatant was collected. The supernatant was filtered through a 0.45 μm cellulose acetate film, and alcohol precipitation was performed using anhydrous ethanol with three times the volume of the supernatant. The resulting precipitate was centrifuged, and 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, and the mixture was shaken for 15 min. The mixture was then centrifuged at 5000 r / min at 4°C for 10 min, the supernatant was collected, and the Sevag treatment step was repeated three times. Each extract was placed in a pre-treated dialysis bag and dialyzed in distilled water for 24 hours, changing the distilled water every 6 hours. After dialyzing, the extract was transferred to a centrifuge tube, frozen in a refrigerator at -80°C, and freeze-dried in a freeze-dryer to obtain Diospyrostrata var. japonica active metabolite polysaccharides.

[0023] Glycosyl group composition analysis was performed on the active metabolite polysaccharides of *Diospyros fusca*. After purification, the polysaccharides were subjected to complete acid hydrolysis and HPLC analysis, and the proportion of monosaccharides in the *Diospyros fusca* biofilm EPS polysaccharides was obtained by comparing them with the peak areas of monosaccharide standards.

[0024] The experimental results are shown in Figure 1 and Table 1. In the glycosyl group composition of the polysaccharides of the active metabolites of *Diospyros kirilowii*, glucose accounts for the largest proportion (43.03%), followed by D-fucose (24.19%), D-xylose (14.82%), D-galactose (12.61%), and D-mannose (12.05%). Gluconic acid, L-rhamnose, and glucosamine account for relatively small proportions.

[0025] [Table 1]

[0026] Example 2 Non-destructive micrometry (NMT, Asahitsuki) uses corresponding microsensors to measure Ca between two target points. 2+ Used to measure concentration gradients. Ca 2+The microsensor has pre-filled 0.5 mM and 0.05 mM Ca 2+ Calibration is performed. After washing the oyster eyespot larvae 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) and allowing them to cure for 20 minutes, the active metabolite polysaccharides of the diatom extracted in Example 1 are added, and Ca 2+ A flux microsensor was placed near the shell edge (approximately 3 μm) to detect the calcium ion exchange status of oyster larvae, and Ca 2+ Flux data was derived from im Fluxes V2.0 software.

[0027] Oyster eyespot larvae in the test solution were used as the control group, and those treated with the test solution to which the active metabolite polysaccharide of *Diospyros fusca* was added were used as the treatment group. Calcium ion flow within the larvae was monitored, and the results are shown in Figures 2 and 3. Under the environment of *Diospyros fusca* active metabolite polysaccharide, oyster larvae showed a clear influx of calcium ions, and the difference was significant.

[0028] Example 3 1. Behavioral statistics were performed on oyster larvae. The results are shown in Figures 4 and 5. From the behavioral data of different groups of oyster eyespot larvae in their attachment environments, when oyster eyespot larvae were placed in an attachment environment for 24 hours, in the group of polysaccharides (also called the polysaccharide group) of active metabolites of the diatomaceous gland, 30% of the larvae were swimming, 64.58% were at the bottom of the polysaccharide group, and 5.42% were crawling, searching for a suitable attachment site. In the control group, 67.59% of the larvae were swimming, and only 32.41% were at the bottom of the attachment site. 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. In the control group, only 1.52% of the larvae underwent metamorphosis. The proportion of swimming larvae (45.00%) was higher than in the polysaccharide group (23.33%), while the proportion of larvae at the bottom (50.91%) was lower than in the polysaccharide group (68.335%). Results from 72h, 120h, and 168h indicate that the overall number of juvenile mollusks in the polysaccharide group steadily increased (15.00%, 30.56%, 51.67%) and was higher than in the control group (5.00%, 14.70%, 29.72%), while the number of swimming larvae steadily decreased (21.67%, 5.00%, 3.33%) and was lower than in 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, compared to only 43.33% (P<0.01) in the control group. This demonstrates that the active metabolite polysaccharides of the diatom can accelerate the metamorphosis process in oyster larvae.

[0029] 2. Oysters were cultured, and when the number of oyster eyespot larvae reached 60%, the active metabolite polysaccharides of the diatom extracted in Example 1 were placed in the larval culture tank at a concentration of 2-5 g / L, and normal aquaculture was carried out, and the metamorphosis rate of the oyster eyespot larvae was detected.

[0030] The experimental results for the active metabolite polysaccharides of the diatomaceous oyster on oyster metamorphosis are shown in Figures 6 and 7. The metamorphosis rate in the control group was 5.3%, while in the diatomaceous oyster active metabolite polysaccharide group it was 16.9%, indicating that the diatomaceous oyster active metabolite polysaccharides significantly improves the rate of attachment metamorphosis in late oyster apical larvae.

[0031] The above embodiments are used solely to illustrate the technical solutions of the present invention and are not limiting. While the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or replace some of their technical features equally, and such modifications or replacements will not depart from the spirit and scope of the technical solutions to which the present invention claims the corresponding technical solutions.

Claims

1. The use of a diatomaceous rhizome active substance for promoting bivalve attachment and metamorphosis, wherein the diatomaceous rhizome active substance is a diatomaceous rhizome active metabolite polysaccharide, which comprises glucose, D-fucose, D-xylose, D-galactose, D-mannose, gluconic acid, L-rhamnose, and glucosamine, and the diatomaceous rhizome active substance causes an influx of calcium ions into oyster larvae, and the bivalve is an oyster. The aforementioned diatomaceous ferox active substance is prepared by a method for preparing a diatomaceous ferox active substance, which includes the steps of: (1) centrifuging diatomaceous ferox in the exponential growth stage, filtering the obtained supernatant, adding anhydrous ethanol, centrifuging again, discarding the supernatant and collecting the precipitate; (2) removing the protein in the precipitate from step (1) with Sevag reagent, centrifuging again and collecting the supernatant; and (3) dialyzing the supernatant from step (2) using a dialysis bag, freezing the dialysate, and then drying it to obtain the diatomaceous ferox active substance. This method is used to promote the attachment and metamorphosis of bivalves.

2. The use of the diatom activating substance according to claim 1 for promoting bivalve attachment and metamorphosis, characterized in that, in step (1), the volume of the anhydrous ethanol is 2 to 5 times that of the supernatant.

3. The use of the diatom active substance according to claim 1 for promoting bivalve attachment and metamorphosis, characterized in that, in step (2) above, the conditions for removing the protein are: shaking temperature: 4-6°C, shaking time: 15 min, and number of repetitions:

3.

4. The use of the diatomaceous diatomaceous activating substance according to claim 1 for promoting bivalve attachment and metamorphosis, characterized in that, in step (3) above, 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.

5. The method of using the diatomaceous ferox active substance, characterized in that when the number of eyespot larvae of bivalves reaches 50% to 60%, the diatomaceous ferox active substance is administered into the larval culture tank, as described in Claim 1, for promoting bivalve attachment and metamorphosis.

6. The use of the diatomaceous ferox active substance according to claim 5 for promoting bivalve attachment and metamorphosis, characterized in that the concentration of the diatomaceous ferox active substance used is 2 to 5 g / L.

Images