JP2008228709A - New microorganism, new microorganism for feed of crassostrea gigas and method for culturing crassostrea gigas by using this new microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new microorganism and the new microorganism for feed of Crassostrea gigas and to provide a method for culturing Crassostrea gigas by using the new microorganism. <P>SOLUTION: There are provided a Pavlova sp. JPCC42D strain (hereinafter referred to as Pavlova sp. JPCC42D), Pavlova sp. JPCC42D used for culture of Crassostrea gigas having ≥1.5 mm shell height and a method for culturing Crassostrea gigas having ≥1.5 mm shell height by using Pavlova sp. JPCC42D alone or a mixture of Pavlova sp. JPCC42D with Chaetoceros calcitrans. The amount of Pavlova sp. JPCC42D occupied in the mixture is preferably 1/4 to 3/4. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) strain, which is a novel microorganism belonging to the order of Pavlova, the new strain for oyster feed, and a method for culturing oysters using the new strain.

In aquaculture of seafood including bivalves, various aquaculture feeds are used depending on the type of fish, but in general, the share of the cost for aquaculture is the highest, with low costs. In order to cultivate seafood, it is important to select and use inexpensive and high growth promoting effects from the very early stage of culturing.
Under such circumstances, in the cultivation of oysters (Grassostrea gigas, hereinafter abbreviated as oysters), diatoms such as Keatoceros calcitrans are commonly used as food (see Patent Document 1), especially oysters. Immediately after hatching, a mixed feed of Kitoceras charis trance and Pavlova tereri (hereinafter abbreviated as Pavlovarteri), which is a kind of marine microalgae, may be used.
JP-A-6-105658

However, even if it cultures using the said feed, the growth promotion effect of a oyster is not fully satisfied, and development of the feed for oyster culture different from the past is desired.
The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel microorganism, the novel microorganism for oyster feed, and a oyster culture method using the novel microorganism.

To solve the above problem,
The invention described in claim 1 is a Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) strain.

  The invention according to claim 2 is an algae belonging to the order Pavlova whose base sequence of 18S rDNA is represented by SEQ ID NO: 1.

  The invention described in claim 3 is a Pavlova sp. JPCC42D strain used for culturing oysters (Grassostrea gigas) having a shell height of 1.5 mm or more.

  The invention according to claim 4 is used for culturing oysters (Grassostrea gigas) having a base sequence of 18S rDNA having a homology of 96% or more with the base sequence represented by SEQ ID NO: 1 and having a shell height of 1.5 mm or more. Algae belonging to the order Pavlova.

  The invention according to claim 5 is a method for cultivating oysters (Grassostrea gigas) having a shell height of 1.5 mm or more using a Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) strain.

  The invention according to claim 6 is a method for cultivating oysters (Grassostrea gigas) having a shell height of 1.5 mm or more using a mixture of Pavlova sp.

  The invention according to claim 7 is characterized in that the amount of Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) occupying in the mixture is 1/4 to 3/4. It is a culture method of (Grassostrea gigas).

  According to the present invention, a new strain of Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) can be provided, and the new strain can be used alone or mixed with Keatoceros calcitrans as feed for oyster culture. As a result, the growth promotion effect of oysters can be enhanced and high quality oysters can be cultivated at low cost.

The present invention will be described in detail below.
The Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) strain of the present invention (hereinafter abbreviated as “Pavlova sp. JPCC42D”) is also found in fresh water and land, but is mainly found as a fine suspended organism in seawater. And as shown in the below-mentioned Example, it obtains by culture | cultivating and singularizing the sample extract | collected from seawater.

  Pavlova SP JPCC42D of the present invention was deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology and was received as receipt number FERM AP-20704 on October 31, 2005. The certificate was not issued for the reason that "contamination due to" was confirmed. At present, the Pavlova SP JPCC42D of the present invention is stored in a state that can be sold at Power Supply Development Co., Ltd.

  Pavlova SP JPCC42D of the present invention has a base sequence represented by SEQ ID NO: 1 as the base sequence of 18S rDNA, as described in Examples below. In addition, the present invention has an algae belonging to Pavlova whose base sequence of 18S rDNA is represented by SEQ ID NO: 1, and has a homology of 96% or more with the base sequence of 18S rDNA represented by SEQ ID NO: 1. , Including algae belonging to the order of Pavlova used for culturing oysters (Grassostrea gigas) with a shell height of 1.5 mm or more.

(Culture properties)
The culture properties of Pavlova SP JPCC42D are as shown below. That is, Pavlova SP JPCC42D cannot be grown in any of broth agar plate culture, broth liquid culture, broth gelatin puncture culture, and litmus milk, and can be cultured in an artificial synthetic medium having the composition shown below. .
Medium composition: sodium nitrate 200 mg / ml, disodium hydrogen phosphate 1.4 mg / l, sodium dihydrogen phosphate 5.0 mg / l, ammonium chloride 68 mg / l, thiamine 0.2 mg / l, biotin 0.0015 mg / l Vitamin B12 0.0015 mg / l, Na ₂ EDTA 37.2 mg / l, FeEDTA 5.2 mg / l, MnEDTA 0.3332 mg / l, Manganese (II) chloride tetrahydrate 0.18 mg / l, Zinc sulfate heptahydrate 0.024 mg / l, cobalt (II) chloride hexahydrate 0.0072 mg / l, molybdate (VI) disodium dihydrate 0.0072 mg / l, copper (II) sulfate pentahydrate 0.0024 mg / L, sodium selenite pentahydrate 0.0016 mg / l, artificial seawater (manufactured by Senju Pharmaceutical Co., Ltd.) 37 g / l Medium Note that the medium of the composition, for example, can also be used agar medium prepared by adding powdered agar 12 g / l.

The pH of the medium is preferably 7.9, and the pH of the medium is adjusted before sterilization. The sterilization conditions of the medium are preferably 121 ° C. and 10 minutes in an autoclave.
Further, Pavlova SP JPCC42D is aerobic, and the culture method is preferably aeration and stirring culture. At this time, the air flow rate is preferably about 2 L / min.
Furthermore, Pavlova SP JPCC42D is a marine microalgae and has light requirements, and therefore light irradiation with an illuminance of about 6000 lux is required during culture. However, a light / dark cycle is not required.
The culture temperature is preferably about 25 ° C., and Pavlova SPJPC42D can be grown well by culturing for 6 days under the above conditions.

Pavlova SP JPCC42D can be subcultured by performing transplanting after culturing for 6 days under the above conditions.
In addition, since storage of Pavlova SP JPCC42D is not suitable for any of the freeze-drying method, the L-drying method and the freezing method (about −80 ° C.), the liquid culture medium having the above composition is used for the interval of about 1 month. Store by performing planting as needed. During storage, light irradiation of about 200 lux is performed by static culture under the above conditions. The storage temperature is preferably about 25 ° C.

  The Pavlova SP JPCC42D of the present invention can be used as a feed in the cultivation of oysters with a shell height of 1.5 mm or more. Here, the shell height refers to the length from the hinge to the tip of the shell of the larvae. That is, Pavlova SP JPCC42D can be suitably used as a feed for larvae of oysters that have passed a predetermined growth period since hatching, not immediately after hatching. The size of the oyster used at this time is not particularly limited as long as it has a shell height of 1.5 mm or more, but preferably has a shell height of 1.5 mm to 6.5 mm. However, the Pavlova SP JPCC42D of the present invention has a high effect of promoting the growth of juvenile shellfish, especially when used as a mixed feed with other marine microalgae as described later, so that the shell height is 1.5 mm. It is more preferable to start feeding as soon as possible.

  Moreover, the feed effect with respect to the oyster of Pavlova SP JPCC42D can be confirmed by the extension of the shell height of the oyster and the increase in the dry weight value. Here, the dry weight value indicates the weight of the oyster including the soft body portion dried under a predetermined condition.

The feeding method of Pavlova SPJPC42D for oysters may be single feeding, but mixed feeding with certain other marine microalgae is preferred.
In the case of mixed feeding, the feed to be mixed is not particularly limited as long as it has a feed effect on oysters, and various marine microalgae can be used, among which Keatoceras calcitrans (Chaetoceros calcitrans) Hereinafter, it is preferably abbreviated as “Ketoceras charistrans”.

  When such a mixed feed is used, the amount of the Pavlova SP JPCC42D of the present invention in the mixed feed is not particularly limited, but preferably 1/4 to 3/4, and 1/2. Is more preferable. Here, “amount” indicates the number of cells. When oysters are cultured using a mixed feed containing 1/2 Pavlova sp. JPCC42D, there is a synergistic effect that promotes the growth of oysters more than when fed Pavlova sp. JPCC42D and the feed to be mixed alone. This is especially effective when the food to be mixed is Keat Seracus charis trance.

  The density of Pavlova SPJPC42D in the aquaculture water when feeding is not particularly limited, and may be appropriately selected according to the density of oysters in the aquaculture water. Specifically, for example, in the case of single feeding, when the density of oysters in the aquaculture water is 25 to 150/5000 ml, the density of the Pavlova SP JPCC42D in the aquaculture water is 3 to 120,000 cells / ml. It is preferable. In the case of mixed feed, the density of the mixed feed may be 3 to 120,000 cells / ml. Therefore, the density of Pavlova SPJPC42D is a value obtained by multiplying the density by the mixing ratio in the mixed feed. Since Pavlova SP JPCC42D is a single-cell algae, the unit of density “cells / ml” is synonymous with “pieces / ml”.

  The number of times the Pavlova SPJPC42D is fed to the oyster is not particularly limited, and may be appropriately adjusted according to the amount of the Pavlova JPJPC42D used for one feeding. For example, when the density of Pavlova SPJPC42D in the aquaculture water at the time of feeding is the above-mentioned preferable value, the number of feedings is sufficient once / day.

  Except for the above points, conventionally known conditions and methods may be applied as conditions for feeding Pavlova SPJPC42D and culture methods for juvenile oysters.

  In general, it is known that the algae of Pavlova is not suitable for long-term storage because it is easily affected by environmental changes and inferior in stability. However, the Pavlova SP JPCC42D of the present invention can maintain a good state by storing it in a cool dark place (for example, 4 ° C.). Therefore, in terms of storage stability, it can be suitably used as a feed for oyster oysters.

  Hereinafter, the present invention will be described in more detail with reference to specific examples. In addition, this invention is not limited to the Example shown below at all.

Example 1
(Acquisition of Pavlova SP JPCC42D)
Sodium nitrate 200 mg / ml, disodium hydrogen phosphate 1.4 mg / l, sodium dihydrogen phosphate 5.0 mg / l, ammonium chloride 68 mg / l, thiamine 0.2 mg / l, biotin 0.0015 mg / l, vitamin B12 0.0015 mg / l, Na ₂ EDTA 37.2 mg / l, FeEDTA 5.2 mg / l, MnEDTA 0.3332 mg / l, Manganese (II) chloride tetrahydrate 0.18 mg / l, Zinc sulfate heptahydrate 0.024 mg / L, cobalt chloride (II) hexahydrate 0.0072 mg / l, disodium molybdate (VI) acid dihydrate 0.0072 mg / l, copper (II) sulfate pentahydrate 0.0024 mg / l, Sodium selenite pentahydrate 0.0016 mg / l, artificial seawater (Senju Pharmaceutical Co., Ltd.) 37 g / l, powder agar 12 / L to the addition of prepared agar medium, in November 2002, was added to the sample 100μl taken from Kitakyushu City Wakamatsu-ku, Wakita coast of sea water the screening was carried out.

(Morphological properties)
As a result of culturing at 25 ° C. for 20 days on the medium, brown marine microalgae colonies having a diameter of about 0.2 mm were obtained. The shape of the colony was punctiform and flat with no bumps. The peripheral edge was the whole edge, and the surface was a smooth shape. The colony was opaque and had a butter-like consistency.
There was no change in colony morphology due to mutation, and no change in colony morphology due to culture conditions or physiological conditions.

The marine microalgae forming the colonies were unicellular algae having a size of 6 to 8 μm, and were yellowish brown and had two flagella flagella and haptonema. Moreover, it had chlorophyll a, c ₁ , c ₂ and c ₃ as plastids, and had a tubular criste-type mitochondria.
From the above properties, the obtained marine microalgae were judged to be haptophyceae algae.
Further, the obtained marine microalgae have unequal length flagella, the long flagella are covered with fine hairs (non-tubular hairs) or humpy scales, and the cells are covered with organic or calcified scales. Not confirmed. These morphological properties are peculiar to the haptophyceae Pavlova algae, and are not found in the other haptophyceae algae, isoclysis, crustacea and primumesium, so Algae were determined to be algae classified as Paplova in the haptophyceae class.

(Base sequence)
From the marine microalgae obtained above, DNA was extracted using a Qiagen kit according to a conventionally known method, and the 18Sr DNA region was amplified by PCR (Polymerase Chain Reaction) method to perform sequence analysis. The base sequence of this region was measured. The base sequence of the obtained 18Sr DNA is shown in SEQ ID NO: 1.

(System analysis)
The obtained 18S rDNA base sequence was collated with the public DNA database Japan DNA Data Bank (DDBJ) to search for homology, and systematic analysis was performed. The resulting phylogenetic tree is shown in FIG.
As a result, the obtained marine microalgae were classified as Pavlova, and were branched separately, so it was determined that they were Pavlova SP JPCC42D, which is a new species of algae.

(Amino acid composition)
Regarding the amino acid composition of Pavlova SPJPC42D, amino acids other than tryptophan were analyzed by an amino acid automatic analysis method, and tryptophan was analyzed by a high-performance liquid chromatographic method, and compared with Pavlovarteri, which is a related species. The results are shown in Table 1.

(Fatty acid composition)
The fatty acid composition of Pavlova SP JPCC42D was analyzed by gas chromatographic method and compared with Pavlovarteri, a related species. The results are shown in Table 2.

(Basic component composition)
The basic component composition of Pavlova SPJPC42D was analyzed by the following method and compared with the related species Pavlovarteri. The results are shown in FIG.
Moisture Normal pressure heating and drying method Crude protein Kjeldahl method Crude fat Diethyl ether extraction method Crude fiber Standing method Crude ash Direct ashing method Soluble nitrogen free Total carotene converted from total balance High-performance liquid chromatography α-carotene High-performance liquid chromatograph Β-carotene high-performance liquid chromatographic method Total ascorbic acid high-performance liquid chromatographic method

(Example 2)
(Confirmation of feed effect of Pavlova SP JPCC42D)
The collected sample used in Example 1 was subjected to static culture for 7 to 10 days using the medium at 25 ° C. to obtain marine microalgae forming brown and green colonies. In order to monomorphize these marine microalgae, inoculate marine microalgae into a liquid medium containing 1 ml of the above-mentioned components, grow by performing stationary culture, and newly proliferate the planted in 1 ml of the liquid medium. Marine microalgae were singulated by repeating the operation of growing in fungi. As a result, Pavlova SP JPCC42D was contained in about 500 obtained marine microalgae. The growth curve of Pavlova SP JPCC42D during culture is shown in FIG.

The resulting Pavlova SP JPCC42D was used as a test feed section, and a diatom commonly used for juvenile culture, kietoceras charistrans (manufactured by Nissin Marine Tech Co., Ltd.) was used as a control feed section, and a comparison of feed effects was performed. .
For the rearing larvae, oyster bottom larvae with a shell height of around 2 to 6 mm were used. The juveniles before the test were stored for about 5 days with no feeding in the state of flowing natural seawater.

  Adjust the temperature of the 8 liter plastic water tank to 5 liters of breeding water with adjusted temperature (here, breeding water is finally filtered through a 1 μm cartridge and then aerated with air filtered through a 0.45 μm cartridge to 23 ° C.) And a filter tray on which 100 juvenile shellfish were placed was accommodated in this water tank. The filter tray on which the larvae were placed was installed so as to be located at the height of the center of the aquarium. In each test feed section and control feed section, three tanks were prepared for each series, and the shell height (growth rate) of the juveniles was calculated from the average value of the juveniles in the three tanks.

  Feed once a day, and measure the cell density in the breeding water of the marine microalgae, which is the feed, in advance of daily feeding, so that the marine microalgae in the breeding water will have a cell density of 50,000 cells / ml. It adjusted and added. Here, the cell density refers to the food density, and this is the same in other embodiments. In addition, from the second day onward, the amount of remaining food in each aquarium was first measured, and after each aquarium was replaced with a washed aquarium, feeding was carried out after total reconstitution with temperature-controlled filtered seawater. The filter tray with juveniles was changed every two days. The larvae were bred for 14 days in a constant temperature breeding room at 23 ° C., and ventilation was not performed during breeding.

  Whether or not the test could be continued depending on the growth status of the larvae was judged by the shell height of the larvae on the 7th day of each test feed zone and the control feed zone.

  The method for measuring and calculating the shell growth rate is as follows. That is, about 30 out of 100 juveniles reared in each water tank, the length from the hinge to the shell tip was measured under a stereomicroscope as the shell height. The measurement was started on the 0th day of breeding, and was performed every 2 days in principle. The increase (%) in average shell height for each breeding day relative to the average shell height on the 0th day of breeding was calculated and evaluated as the shell height growth rate.

The high shell growth rate up to the last day of the test was 25.4 ± 4.0% in the Keatceras charistrans test feed group and 18.6 ± 3.9% in the Pavlova SPJPC42D test feed group.
There was no significant difference in the shell growth rate between Keat Seracus charis trance and Pavlova sp. JPCC42D. Therefore, Pavlova sp. JPCC42D has the same level of feed effect on juveniles as Keat Selas charis trance. It was confirmed.

(Comparative Example 1)
Among about 500 marine microalgae obtained from seawater by the method described in Example 2, Primenesium nemetechum, Nannochloris maculeate, Pavlova gilance (Pavlova gyrans test) As a feed section, the effect of the feed was compared in the same manner as in Example 2 with Keat Seracus charistrans (manufactured by Nissin Marine Tech Co., Ltd.) as the control feed section.
As a result, none of the strains in the test food section was able to contribute to the growth of juvenile shellfish, and no feed effect was shown.

(Example 3)
(Confirmation of palatability of oyster oysters to Pavlova SPJPC42D)
Paglova sp. JPPCC42D obtained in Example 2 and transformed into a single fungus was used as a test feed section, and kieselguhr commonly used for larval shellfish culture, Keat Seros charis trance (manufactured by Nissin Marine Tech Co., Ltd.) as a control feed section. We compared the taste of juvenile shellfish.

  For the rearing larvae, oyster bottom larvae with a shell height of around 2 to 6 mm were used. The juveniles before the test were stored for about 5 days with no feeding in the state of flowing natural seawater.

  Adjust the temperature of the 8 liter plastic water tank to 5 liters of breeding water with adjusted temperature (here, breeding water is finally filtered through a 1 μm cartridge and then aerated with air filtered through a 0.45 μm cartridge to 23 ° C.) The filter tray on which 40 juvenile shellfish were placed was accommodated in this water tank. The filter tray on which the larvae were placed was installed so as to be located at the height of the center of the aquarium. In each test feed section and control feed section, three tanks were prepared for each series, and the shell height (growth rate) of the juveniles was calculated from the average value of the juveniles in the three tanks.

  Feed once a day, and measure the cell density of marine microalgae, which is the feed, in advance of daily feeding, and adjust the marine microalgae to the breeding water so that the cell density is 100,000 cells / ml. Added. From the second day onward, each breeding aquarium was replaced with a washed aquarium, and then feeding was performed after the total water was changed with temperature-adjusted filtered seawater. The filter tray with juveniles was changed every two days. The larvae were bred by the above operation for 10 days in a constant temperature breeding room at 23 ° C., and the breeding water was not vented during the breeding.

  In order to grasp the decreasing process of the feed density in the breeding water with the passage of breeding time from 0 to 24 hours, the feed density in the aquarium after adjusting to the target density was measured. In order to confirm the change in the feeding amount of the juvenile shellfish over time, the remaining amount of feeding was measured after 24 hours and after the second day. Sampling of the sample was carried out after sufficiently stirring the breeding water in the aquarium. Marine microalgae, which are feeds, did not show nonspecific adsorption to the inner walls of the rearing tank or the equipment in the tank during the rearing of juveniles, and the decrease in feed density was due to the feeding being taken up by the rearing juveniles It was thought that.

  The method for measuring the amount of remaining food is as follows. That is, (1) A Pasteur pipette was used for sampling a sample, and the sample was randomly sampled 3 to 5 times from the water tank and collected in one Eppendorf tube. (2) When the marine microalgae as a feed was mobilized, 1% formalin was added at 1% per volume to stop the movement. (3) For the sampled sample in the Eppendorf tube, the amount of remaining food was measured three times and the average value was used. However, when the measurement result has a large error, the measurement was performed again to increase the measurement accuracy.

  The ratio of the obtained residual feed to the feed at the start of breeding is calculated as the residual feed rate, and the change over time in the residual feed rate is plotted in a graph in FIG. From FIG. 2, the decrease in feed density due to feeding of juvenile larvae after feeding was not confirmed as a significant difference between the Keatseras charis trance as the control feed zone and the Pavlova SPJPC42D as the test feed zone. It was confirmed that juvenile shellfish showed the same degree of palatability as Peat Blossom SP JPCC42D in comparison with Keat Seracus Charis trance.

Example 4
(Confirmation of stability of Pavlova SP JPCC42D)
Generally, algae of Pavlova is not suitable as a feed because it is easily affected by environmental changes due to transportation or the like and has poor stability. Therefore, the stability of Pavlova SP JPCC42D was confirmed by the following method. That is, using the Pavlova SP JPCC42D obtained in Example 2 as a single fungus, as a culture transfer condition, aeration culture in a 200 ml Erlenmeyer flask was conducted for 7 days, aeration culture in a 500 ml flat flask was conducted for 4 days, and aeration culture in a 10 l bottle. For 5 days to obtain a culture solution having a cell density of 5 to 10 million cells / ml. Centrifugation is performed using 1 lOg of the culture solution at 11000G for 10 minutes, the cell density is concentrated to 100 million cells / ml, and the concentrated cell fluid is stored in a cool dark place (4 ° C) for 10 days. The cell density during this period was measured. The results are shown in FIG.

  As shown in FIG. 3, Pavlova SP JPCC42D is sufficient as a food because even when stored in a cool and dark place (4 ° C.) for 10 days, the cell density can be maintained in a good state with almost no decrease in cell density during this period. It was confirmed that it has a good stability.

(Example 5)
(Single feeding and mixed feeding tests of Pavlova SPJPC42D for oyster oysters)
Pavlova SPJPCC42D obtained in Example 1 and transformed into a single fungus, and Pavlova territus, a related species of Pavlova SPJCC42D, and Keat Serras charistrans (manufactured by Nisshin Marine Tech Co., Ltd.), a diatom commonly used in juvenile culture ) Was used to carry out a single feeding test and a mixed feeding test on oyster oysters.

  The test plots are the Keatseras charistrans test zone, the Pavlovarteri test zone, the Pavlova SPJPCC42D test zone as the single feeding test zone, and the 50% Keatseras charistrans + 50% pavlovarelli test zone as the mixed feeding test zone in which each feed is mixed. Five sets were established:% Keat Seras Charis Trans + 50% Pavlova SP JPCC42D Test Zone. Here, “%” refers to “cell number (concentration)%”.

  For the rearing larvae, oyster bottom larvae with a shell height of around 2 to 6 mm were used. The juveniles before the test were stored for about 5 days with no feeding in the state of flowing natural seawater.

  Adjust the temperature of the 8 liter plastic water tank to 5 liters of breeding water with adjusted temperature (here, breeding water is finally filtered through a 1 μm cartridge and then aerated with air filtered through a 0.45 μm cartridge to 23 ° C.) The filter tray on which 40 juvenile shellfish were placed was accommodated in this water tank. The filter tray on which the larvae were placed was installed so as to be located at the height of the center of the aquarium. In each test feed section and control feed section, three tanks were prepared for each series, and the shell height (growth rate) of the juveniles was calculated from the average value of the juveniles in the three tanks.

  Feed once a day, and measure the cell density of marine microalgae, which is the feed, in advance of daily feeding, and adjust the marine microalgae to the breeding water so that the cell density is 100,000 cells / ml. Added. From the second day onward, each breeding aquarium was replaced with a washed aquarium, and then feeding was performed after the total water was changed with temperature-adjusted filtered seawater. The filter tray with juveniles was changed every two days. The larvae were bred by the above operation for 10 days in a constant temperature breeding room at 23 ° C., and the breeding water was not ventilated during the breeding.

  Whether or not the test could be continued depending on the growth status of the larvae was judged by the growth rate of the larvae on the 7th day of each test feed and control feed.

First, the survival rate of juveniles for single feeding and mixed feeding was confirmed.
The method for measuring and calculating the survival rate of juvenile shellfish is as follows. That is, individuals who died by opening at the time of shell height measurement were removed, and the number of individuals was recorded in each tank. Then, using this numerical value, the ratio of the number of surviving individuals to the number of individuals at the start of breeding was calculated and used as the survival rate.

  As a result, the survival rate in each water tank up to the last day of the test was in the range of 88 to 100%, and no difference was observed in the survival rate between the feeds.

Next, the preference of juveniles for single feeding and mixed feeding was confirmed.
In order to grasp the decreasing process of the feed density in the breeding water with the passage of breeding time from 0 to 24 hours, the feed density in the aquarium after adjusting to the target density was measured. In order to confirm the change in the feeding amount of the juvenile shellfish over time, the remaining amount of feeding was measured after 24 hours and after the second day. Sampling of the sample was carried out after sufficiently stirring the breeding water in the aquarium. Marine microalgae, which are feeds, did not show nonspecific adsorption to the inner walls of the rearing tank or the equipment in the tank during the rearing of juveniles, and the decrease in feed density was due to the feeding being taken up by the rearing juveniles It was thought that.

  The method for measuring the amount of remaining food is as follows. That is, (1) A Pasteur pipette was used for sampling a sample, and the sample was randomly sampled 3 to 5 times from the water tank and collected in one Eppendorf tube. (2) When the marine microalgae as a feed was mobilized, 1% formalin was added at 1% per volume to stop the movement. (3) For the sampled sample in the Eppendorf tube, the amount of remaining food was measured three times and the average value was used. However, when the measurement result has a large error, the measurement was performed again to increase the measurement accuracy.

The ratio of the obtained residual feed to the feed at the start of breeding is calculated as the residual feed rate, and the change over time in the residual feed rate is plotted in a graph in FIG. As shown in FIG. 4, the decrease in the residual feed rate due to the feeding of the juvenile larvae after feeding did not show a large difference between the Keatseras charistrans and Pavlova SP JPCC42D when compared in the single feeding test section. However, in Pavlovarteri, it was shown that the residual feed rate was higher than that of the two single feeding test areas, that is, the amount of food intake was low.
On the other hand, there was no significant difference in the residual feed rate between the mixed feeding test sections.

  From the above, juveniles show the same degree of preference as the Keat Seracus charis trance for Pavlova sp. JPCC42D in single feeding, and 50% Keat Selas charis trance + 50% Pavlovarteri and 50% Keat Selas charistrans in mixed feeding. + 50% Pavlova SP JPCC42D showed similar palatability.

Subsequently, the feeding effect on single shells and mixed feed juveniles was confirmed.
The method for measuring and calculating the shell growth rate is as follows. That is, for all 40 juveniles reared in each water tank, the length from the hinge to the shell tip was measured under a stereomicroscope as the shell height. The measurement was started on the 0th day of breeding, and was performed every 2 days in principle. The increase (%) in average shell height for each breeding day relative to the average shell height on the 0th day of breeding was calculated and evaluated as the shell height growth rate. The results are shown in FIG.

From Fig. 5, the shell growth rate up to the last day of the test is 24.3 ± 1.9% in the Keatseras charistrans test group and -0.3 ± 1.9% in the Pavlovartelli test group. It was 19.3 ± 4.0% in the Pavlova SP JPCC42D test section. In other words, the Keat Serras Charis Trans test zone and the Pavlova SP JPCC42D test zone showed a shell height growth rate of about 20%, but the Pavlovarteri test zone showed that the shell height of the juveniles could not be grown at all. .
In addition, there is no significant difference in the shell growth rate between the Keat Seras charistrans test zone and the Pavlova SP Japan JPCC42D test zone. Was confirmed.

  On the other hand, in the mixed feeding test group, 26.3 ± 3.0% in the 50% Keat Serras charis trance + 50% Pavlovarteri test group, and 52.2 ± 8.9% in the 50% Keat Serras charis trance + 50% Pavlova SPJPC42D test group. It was shown.

The shell growth rate of the 50% Keat Seras charis trance + 50% Pavlovartelli test plot is 2.2 times the average value of the shell growth rate of each single feed test plot. It was confirmed to have.
On the other hand, the shell growth rate of the 50% Keat Seracus charis trance + 50% Pavlova SP JPCC42D test plot is 2.4 times the average value of the shell growth rate of each single feeding test plot. The effect is higher than that of the 50% Keat Seracus charistrans + 50% Pavlovarteri test area, and the shell growth rate is extremely high at 52.2 ± 8.9%. It was confirmed that the mixture of Charistrans and Pavlova SP JPCC42D is extremely excellent as a feed for oysters.

Subsequently, the rate of increase in dry weight of juveniles with respect to single feeding and mixed feeding was confirmed.
The dry weight value measurement and calculation method is as follows. That is, the surface of the juvenile after completion of the breeding was washed with demineralized water and then placed on a glass petri dish, and the juvenile was dried at a dryer temperature of 60 ° C. until an impact was obtained. After drying, the total weight of the juvenile shellfish was measured with a scale, and the dry weight per juvenile shellfish was calculated by dividing by the number of individuals housed in the petri dish. Here, the dry weight value indicates the weight of the young shellfish including the soft body part. Furthermore, the increase rate of the dry weight value after completion | finish of breeding with respect to the dry weight value before breeding was calculated, and what was plotted on the graph is shown in FIG.

From Fig. 6, the dry weight increase rate was 65.8 ± 27.8% in the Keat Sera charis trans test group, -11.7 ± 6.7% in the Pavlovartelli test group, and the Pavlova SPJ JPC42D test. It was 59.8 ± 21.3% in the section. In other words, the dry weight increase rate of about 60% was shown in the Keat Seracus charistrans test zone and the Pavlova SP JPCC42D test zone, but the Pavlovarteri test zone showed no food effect including the soft body part, and the growth of the larvae was completely absent. Results showed that they could not contribute.
In addition, there was no significant difference in the rate of increase in dry weight between the Quitoceras charistrans test group and the Pavlova SP Japan JPCC42D test group, and there was an equivalent feed effect on the growth of juveniles including the soft body. It was confirmed that

  On the other hand, in the mixed feeding test group, 54.1 ± 11.8% in the 50% Keat Serras charis trance + 50% Pavlovarteri test group, and 177.3 ± 29.3% in the 50% Keat Serras charis trance + 50% Pavlova SPJPC42D test group. It was shown.

The dry weight increase rate of the 50% Keat Seras charistrans + 50% Pavlovartelli test plot is 2.0 times the average dry weight gain of each single feed test plot. It was confirmed to have.
On the other hand, the dry weight increase rate of the 50% Keat Seracus charis trance + 50% Pavlova SP JPCC42D test section is 2.8 times the average value of the shell high growth rate of each single feeding test section, and mixed feeding The effect is higher than that of the 50% Keat Seracus charistrans + 50% Pavlovarteri test zone, and the shell growth rate is extremely high at 177.3 ± 29.3%. It was confirmed that the mixture of Charistrans and Pavlova SP JPCC42D is useful for the growth of juveniles including soft bodies and is extremely excellent as a feed for oysters.

  From the above, it was confirmed that Pavlova SP JPCC42D of the present invention has excellent storage stability and is useful as a feed for oyster oysters, and that high quality oysters can be produced by the cultivation method of the present invention using Pavlova SP JPCC42D. .

  According to the present invention, oysters can be cultivated in a shorter period of time and at a lower cost than before, so that the present invention is useful in the fishery industry, and can supply inexpensive and high-quality oysters.

It is a graph which shows the growth curve of Pavlova SP JPCC42D in Example 2. It is a graph which shows the test result of the palatability with respect to Pavlova SPJPCC42D of the oyster larva in Example 3. It is a graph which shows the test result of stability of Pavlova SP JPCC42D in Example 4. It is a graph which shows the test result of the palatability of the oyster oyster with respect to the single feeding and mixed feeding of Pavlova SP JPCC42D in Example 5. In Example 5, it is a graph which shows the feed effect of the single feeding and mixed feeding of Pavlova SPJPC42D with respect to the oyster oyster. It is a graph which shows the result of the dry weight increase of the oyster juvenile by the single feeding of Pavlova SP JPCC42D in Example 5, and mixed feeding. It is a phylogenetic tree which shows a system analysis result of Pavlova SP JPCC42D.

Claims (7)

  Pavlova SP JPCC42D (Pavlova sp. JPCC42D) strain.   An algae belonging to the order of Pavlova whose base sequence of 18S rDNA is represented by SEQ ID NO: 1.   Pavlova sp. JPCC42D strain used for culturing oysters (Grassostrea gigas) with a shell height of 1.5 mm or more.   An algae belonging to the order of Pavlova which is used for cultivation of oysters (Grassostrea gigas) having a base sequence of 18S rDNA having a homology of 96% or more with the base sequence represented by SEQ ID NO: 1 and having a shell height of 1.5 mm or more.   A culture method of oysters (Grassostrea gigas) having a shell height of 1.5 mm or more using a Pavlova SPJPC42D (Pavlova sp. JPCC42D) strain.   A culture method for oysters (Grassostrea gigas) having a shell height of 1.5 mm or more using a mixture of Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) strain and Keatoceros calistrans.   The culture method of oysters (Grassostrea gigas) having a shell height of 1.5 mm or more according to claim 4, wherein the amount of Pavlova sp. JPCC42D (Pavlova sp. JPCC42D) strain in the mixture is 1/4 to 3/4.

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