초록 ▼

○ 연구결과
□ 건강한 바지락 모패 발굴을 위한 연구
-. 우리나라 전 연안에 서식하는 바지락의 번식량, 번식주기 및 기생충감염도 조사
-. 바지락 질병감염 및 병원체에 대한 면역력 측정 기술 개발
-. 바지락 집단 구별을 EST-linked microsatellite marker 개발 및 이를 이용한 바지락 집단간 유전적 특성 비교
-. 바지락과 아기바지락의 유전학적 특성 비교 (48개의 유전정보 GenBank 등록)
□ 바지락 종묘의 안정적인 공급에 관한 연구
- . 순환여과 시스템을 이용한

○ 연구결과
□ 건강한 바지락 모패 발굴을 위한 연구
-. 우리나라 전 연안에 서식하는 바지락의 번식량, 번식주기 및 기생충감염도 조사
-. 바지락 질병감염 및 병원체에 대한 면역력 측정 기술 개발
-. 바지락 집단 구별을 EST-linked microsatellite marker 개발 및 이를 이용한 바지락 집단간 유전적 특성 비교
-. 바지락과 아기바지락의 유전학적 특성 비교 (48개의 유전정보 GenBank 등록)
□ 바지락 종묘의 안정적인 공급에 관한 연구
- . 순환여과 시스템을 이용한 바지락의 성 성숙 유도
- . 바지락 인공산란을 통한 바지락 유생 발달 관찰 및 유생 사육
- . 바지락 인공종묘 생산
- . 유생발달 단계에 따른 채묘법 개발
- . 바지락 먹이생물 확보 및 대량배양법 개발
□ 바지락 중간 육성에 관한 연구
- 바닥식 양성법 과 수하식 양성법을 이용한 바지락 성장 비교

Abstract ▼

Chapter 1. The development of mass production systems for Manila clam (Ruditapes philippinarum) seed
The Manila clam or short-neck clam (Ruditapes philippinarum) is an important aquaculture species including oyster that inhabits on sandy or muddy tidal flats along the coastal areas in Korea. In 1

Chapter 1. The development of mass production systems for Manila clam (Ruditapes philippinarum) seed
The Manila clam or short-neck clam (Ruditapes philippinarum) is an important aquaculture species including oyster that inhabits on sandy or muddy tidal flats along the coastal areas in Korea. In 1990 Korea produced 70,000 tones of clams and the clam landing declined gradually.
Currently 30,000-40,000 tons of clams are produced along the west and south coast of Korea. The natural clam seed supply for clam industry has experienced the decline in the seed harvest, due to decline in clam culture area by massive development and reclamation, deterioration of habitat quality, increase in infectious diseases and over-fishing. To compromise shortage of the seed supply, Korean clam industry has imported clam spats from oversea, mainly from China, and the Korean clam industry has paid over6 billion won for the imported seeds. The imported clam seeds are often exhousted during long-distant transportation resulting in depressed viability and subsequent mortality after sowing in the clam beds. Accordingly, developing a mass production of clam seed is greatly demanded by the clam industry of Korea and the technique would improve the seed supply condition.
Chapter 2. Aim and scope of the study
To obtain a high quality broodstock for the seed production, 1) we investigated the reproductive effort, condition index, gametogenic condition, parasite infection and immune capacity of Manila clams collected from the west, south and east coast and from Jeju Island over 3 year periods. 2) We examined the growth and overall pathogenic condition of different age of Manila clam raised in intertidal clam culture grounds. 3) To identify genetic characteristics of clam populations in Korea, we developed genetic markers based on EST-linked microsatellite and confirmed the marker polymorphism among different clam populations. Additionally, the polymorphic markers were utilized in discrimination of natural native Korean clam populations and the clams originated from overseas. 4) We studied genetic characteristics of two species of Ruditapes (R. philippinarum and R. variegatus) clams inhabiting in Jeju Island and morphological characteristics.
In an attempt to achieve stable clam seed production, 1) Manila clams collected in early spring were conditioned using indoor recirculation system through controlling of water temperature and food supply. In addition, reproductively mature adults clams were induced to spawn and subsequent larval development was observed using scanning electron microscopy and compound microscope. 2) To develop massive seed harvest technique in hatchery condition, larval settlement and subsequent collection method was developed by eliminating harmful organisms in the tank during the larva development. 3) To optimize the larval growth in the tank, we examined the supply of algal food to different size of clam larvae and spats. 4) A suspended culture technique using net-bag was developed and growth rate and parasite load in clams in suspended culture and bottom culture were compared. Growth rate of clam in suspended culture was 2.5 times faster with much lower level of the parasite infection.
Chapter 3. Research method and result
1. Evaluation of healthy Manila clam broodstock
a. Evaluation of reproductive effort, condition index and P. olseni infection intensity of Manila clam on the East, West and South coasts and Jeju Island, Korea in 2009, 2010 and 2011.
The reproductive condition and reproductive effort have been considered to be an important element to select quality broodstock for artificial seed production. To obtain optimal and quality broodstock clam, reproductive effort, condition index and parasite infection intensity, clams were collected from 25 sites on the East, West, South coasts and Jeju Island in June or July in 2009, 2010 and 2011. The condition index (CI) was found to be highest in clams from the west coast.
In particular, clams from Duruini, Hwangdo, and Boryoung showed fairly higher CI. In June and July, reproductive condition of clams on the south coast was partially spawned while clams from the west coast such as Sunjae, Sungam, Gonam and Imwon were in ripe. This finding suggested that when clams are fully matured in June, it is suitable period to reserve large adult clams used as broodstock in hatchery operation. GSI and fecundity of clam was higher in June in 2009 and 2011, while it was somewhat lower in July in 2010. Infection intensity of P. olseni in clams was found to be higher on the south coast followed by west coast. The parasite burden was much lower among clams from Jeju Island and from the east coast. In conclusion, clams from Bangpo and Padori in Chungnam, Geojae in Kyongnam province were considered to be a useful broodstock for artificial seed production, which showed higher CI and low parasite infection level.
b. Immuno-capacity and resistance to infection of clams from different coastal regions
b-1 Health assessment and monitoring growth and parasite infection of clams in different age group
b-1-1. Evaluation of health level of clams to secure healthy brood stock
To obtain quality clams to be used as broodstock in the seed production, clam must have superior physiological characteristics and a technique to identify such superior characteristics must be developed. Immune function of Manila clam was assessed in this study using flow cytometry and we compare immune function of clams in field and at laboratory after transportation to the laboratory. The immune parameters including hemocyte viablity phagocytosis rate and the cell DNA damage level were found to be much better in clams before transported to the laboratory, suggesting that assessment of the immune parameters is a good technique to locate healthy broodstock.
b-1-2. Age-dependent growth P. olseni infection intensity in bottom cultured clam
Growth, P. olseni infection intensity and mortality of various age groups including spat, 1-year old and 2-year old manila clams were examined in this study. Clams used in the experiment were installed in cages and raised in intertidal mud flat. The clams cultured on tidal flat showed age-dependent shell growth, P. olseni infection intensity, and mortality rate. The shell growth rate was found to be highest among 1 year old clams and the shell growth rate decreased as the age increase. Infection intensity of P. olseni was the lowest in spats and the infection rate increased as the clam age become higher. The mortality rate was also found to be lower in juvenile clams.
b-2 Physiological response of clam populations challenged by P. olseni
Clam populations from Gangjin (Jeonnam Province), Hongseong (Chungnam Province) and Jeju were challenged with the portozoan parasite, P. olseni and the immune reaction were compared using flow cytometry. The challenged 3 populations by parasitic and bacterial pathogens were examined in terms of presence or absence of resistance to the infection to compare different immune responses and viabilities. As a result, the immune parameters of clam from Gangjin was significantly higher than that of other groups and its mortality rate induced by the pathogen challenge was comparatively lower. It was believed that Gangjin clam has physiologically superior characteristics compared to other clams in other areas, and it suggested presence of pathogen-resistant clam strains in nature.
C. Genetic characterization of Manila clam in Korean waters
c-1 Development of microsatellite maker to differentiate Manila clam populations
In the present study, we developed EST-derived microsatellite markers to investigate genetic characteristics of Manila clam populations in Korean waters, and to identify the marker polymorphisms in Manila clam populations. From the 1,128 unique genes (Expressed sequence tags, EST) originated from Manila clam hemocyte cDNA library constructed in 2006, we discovered 11 microsatellite candidate clones including over 5 repeats of di, tri, tetra, hexa, penta nucleotides after microsatellite screening, and examined the PCR amplification for marker optimization. Eleven verified markers were determined the polymorphism in 15 clams. Characteristics of 11 microsatellite loci are as followed; the numbers of alleles per locus ranged from 3 to 11 with an average 6.5. The observed heterozygosity was 0.0667 to 0.7692, while expected heterozygosity was 0.3571 to 0.8843. Also, 6 markers (KR1,6,11,12,14,17,19) were not amplified in some individuals, indicating that the null alleles are present in the Manila clam. The EST-linked microsatellites used in this study were found to be a useful resource for population genetic of R. philippinarum.
c-2 Genetic characterization of Manila clam populations between Korea and foreign country using microsatellite marker
To compare genetic variation of Manila clam populations in Korea and clams in other countries, we sampled clams from 8 sites on the west coast of Korea (Hwangdo, Boryoung, Gomso, Geojae, Imwon, Sungsan, Seogwi and Oeido), 2 sites in China (Xiamen and Yentai), Japan (Kanazawa-ku) and Hong Kong. The genotyping of collected clams was carried out using 5 markers (KR2,6,14,17,19) and then analyzed the genetic distance and phylogeny. As a result, the genetic distance and phylogeny of 5 markers (KR2,6,14,17,19) showed that Boryoung (Korea) and Yentai (Xiamen) formed a single clade with closet relationship than any other populations. Oeido (OD) and Hongkong (HK) also distinguished as a group (R. variegatus) of the genus Ruditapes from Manila clam populations. This finding suggested that the EST-linked microsatellites used in this study were found to be a useful resource for population genetic of R. philippinarum as well as cross-species amplification of the genus Ruditapes.
c-3 Comparison of genetic characteristics of Manila clam (R. philippinarum) and variegated carpet shell (R. variegatus)
In the national survey of Manila clam, we found the two species of clam, Ruditapes philippinarum and Ruditapes variegatus inhabiting in Jeju Island. Consequently, we isolated three nuclear (18S rDNA, H3 and ITS) and one mitochondrial DNA (COI) from R. philippinarum and R. variegatus collected from coastal Jeju to examine taxonomic affiliation of the two species. The sequence comparison indicated that the two populations of clams sampled from sand flats in Sungsan and Seogwipo were identified as R. philippinarum, while the other two populations sampled in pebbles beach in Oeido and Geumneung were identified as R. variegatus. The ratio of depression of a pallial sinus to shell length in R. philippinarum was a significantly high compared to that of R. variegatus (p<0.05). In addition, the pair-wise genetic distance and phylogenetic analysis of the 2 clam populations formed a monophyletic clade with published sequences of other population of R. philippinarum as well as R. variegatus. The morphology as well as the genetic analysis carried out in this study indicates that R. philippinarum and R. variegatus are genetically distinct and different in the genus Ruditapes.
2. Study on Manila clam seed production in hatchery
a. Conditioning of clam broodstock in an indoor recirculation system
In an attempt to produce clam seed in an hatchery condition, we have conditioned the clam broodstock in an indoor recirculation system. Manila clams to be used as broodstocks were collected from Hongsung on the west coast of Korea in April 2010, when the water temperature remained 13°C. To condition the broodstock, water temperature in the indoor tank was raised from 13 to 20°C by elevating the water temperature daily by 1°C for 7 days. The clams were fed with concentrated algal paste, Shellfish Diet (Reed Mariculture, USA) by 3% of the clam dry tissue weight twice a day. To monitor the reproductive condition, 20 clams were sampled weekly for 57 days. Level of gonad development was determined using histology and quantity of eggs produced was assessed by the clam egg-specific antibody in ELISA. During the course of experiment, mortality remained as 1.1%. Histology revealed that reproductive condition of clams at the beginning of the experiment was early development. Reproductively ripe and ready for spawning clam was first appeared on day 42. At the end of study on day 57, 40% of clams in conditioning became reproductively ripe and ready for spawning. On day 22, the GSI was estimated to be 3.7% and it was 8.2% on day 57. The estimated fecundity of ripe clams ranged 2,876,684±1,473,906 eggs/clam. The protozoan parasite Perkinsus olseni infection intensity in clam also increased during the condition period, from 272,836±99,844 cells/clam to 705,064±313,231,844 cells/clam at the end of the experiment. Relatively small amount of egg produced at the end of the experiment was in part, explained by the high level of parasite infection.
b. Spawning induction and larval development in the Manila clam
To understand hatching and subseqeunt larval development, sexually mature clams were induced to spawn in indoor tanks and the larvae were raised by supplying microalgae produced in the laboratory. Manila clams were collected from Kymnyeong in Jeju Island in August, 2011 and maintained in an indoor tank with supply of cultured microalgae. The clams from Kymnyeong spawned in the tank and produced approximately 5 million eggs per clam. Using compound microscope and scanning electron microscopy (SEM), the embryo and larval development of clam were recorded. The size of fertilized egg ranged from 55 to 60 um, and the egg formed polar body within 1 hr. After progression in the cell division through 2, 4, and 8 cell stages, the gastrula stage of embryo observed in 5 hrs of post-fertilization and initiated the free swimming with developing the protoconch. At 19 hrs in post-fertilization, the embryos reached the D-shape stage and showed feeding behavior. The larvae were fed with micro-algae, Isochrysis galbana and Chaertoceros gracilis and these algae could be observed in the digestive rack of the embryos. At 2 day, the embryos developed into the early D-shaped larvae with velum. As passed through four steps of development such as straight hinge veiliger, early umboned veliger, late umboned veliger and pediveliger, the embryos was reached post-larvae stage with siphon at 36 days. During the period of experiment, the daily growth rate of clam larvae was 4.6 μm and it reached 281.2 μm in post setting.
3. Mass production of R. philippinarum seed in hatchery
a. Seed production system for Manila clam spat
To produce clam seeds in hatchery, 3~5 years old adult clams were selectively chosen as broodstocks. The adult clams were rinsed with 3 ppm KMnO4 to eliminate parasites and pathogens. After transferred to the hatching pond, the water was changed everyday and food organisms were supplied 4 times a day after checking the decrease of the food level. After examining reproductive condition of the broodstock clams, they were transferred to the hatching tank and spawning was induced by 1) 4~5 times of desiccation stimulus, 2) freshwater stimulation (30 minutes of immersion), and 3) cold temperature stimulation (30 minutes in the refrigerator). Just prior to spawning induction, 3 ppm EDTA was added in the hatching pond, one capsule (0.1 g) of vitamin was also added. Spawning starts between mid-night and 2 a.m. under dark and quiet conditions. The water temperature of the hatching pond was adjusted to 23°C, then the plate was transferred to another hatching pond after the density of the fertilized egg reached 15~20 ind./mL. After 24 hours after fertilization D-larvae stage was observed.
Food organism was provided at 6 hour interval for 5~6 days, and water was changed twice a day. After 2 weeks, Manila clam larvae metamorphosed through umbone stage and reached 200~300 μm in size and settled down on the pond floor. After the settlement, larvae are transferred to a larvae nursery pond, water circulation and food supply were kept in same condition, and transferred to other pond after screening with different size using Muller gauze.
b. Seed collecting technique for the mass production
Since clam larvae in D-shape larval stage need to be transferred with seawater, 60 μm Muller gauze was placed on a bucket and the seawater was withdrawn using siphon. The clam larvae hold in the bucket were transferred other water tanks. When the larvae settled after umbone stage, they were transferred to the larvae nursery pond, which was covered with mud to assist the settling and secure habitats for the larvae. Finally, the mud and waste material in the pond were discarded with dead larvae and the slow growing larvae were also eliminated. Fine mud filtered through 60 μm net was supplied into the larvae nursery pond, and spats were filtered onto 100, 200, 250, 320, or 420 μm Muller gauze according to the size. Because the water level of the settling pond was lower than the larvae nursery pond, Muller gauze was placed at the discharge pipe and filtered seawater was sprayed onto the pond wall to prevent dry. All the mud and spats were completely discharged slowly and filtered to harvest the clam spats. The clam larvae harvested from nursery pond were filtered onto 500 μm mesh and weighed, then wet towels with filtered seawater was placed underneath the transportation container and transported
4. Mass culture techniques for the food organism of Manila clam
a. Mass culture techniques of microalgae as food for clam spats
One of the most important elements in the seed production in hatchery condition is the mass culture of microalgae as feed and pure culture of different size of algae to feed the different size of larvae or spats. To produce mass amount of microalgae, several stages of culture were performed according to the size of the culture vessel, and inexpensive nutrients were supplied in the media to secure the economy of the culture operationa. Microalgae selected as food organisms in this study included Chaetoceros mulleri, Dicrateria inornata, Isochrysis galbana, Ｎitzschia sp., Platymonas subcordiformis, and Tetraselmis suecica.
1st stage: stock culture (250 mL, 500 mL) → 2nd stage: small scale culture (2L, 3L) → 3rd stage: extended culture (middle culture; 20L) → 4th stage: extended culture (60L) → 5th stage: mass culture (6 m³)
Culture stages were composed of 4 to 5 sub culture steps with increasing volume, and the maintenance of stocks were carried out at a clean room at Gwangju University under the constant temperature. The concentration of algae before the inoculation of each culture stage was performed when Tetraselmis suecica and Platymonas subcordiformis reached 200,000 cells/mL, and Ｎitzschia sp., Isochrysis galbana, Chaetoceros mulleri became 5,000,000 cells/mL. Before inoculation, the physiological status of phytoplankton was checked finally with microscope and fluorometer whether the FRI values were above 0.6.
b. Improvements of culture media and mass culture or food organism
The primary purpose of microalgae culture study was to develop low cost culture media to overcome the difficulties involved in algae mass culture and to maximize the benefit. Inexpensive agricultural fertilizer was tried in this study to enhance the algae growth in the media. However, it was realized that the agriculture fertilizer as nutrients in the algae culture had difficulty to reach the peak within a short period (5 days) and can not be used for an extended period, due to the imbalance in nutritional contents. Recipe for the mass culture of micro algae in the water tanks includes.
c. Concentration system for the food organism
Food organisms were cultured in massive volume using a outdoor mariculture farming facility and concentrated to produce almost limitless amount. Because the microalgae were culture under a natural conditions, it was expected that the algae produced from this study could be healthier than the algae produced indoor incubation facility. Before condensing the algae, it was essential to remove impurities in the media including suspended solids and zooplankters such copepods.
At 1st stage, the cultured seawater was passed through the long-hair filtration system, at 2nd stage, cloth bag filter removes 50~80 μm sized particles, at 3rd stage, the cartrodge filter removed 20~30 μm sized particles. To reduce effect of high temperature, a cooling system was installed. The concentration system was a modular structure adopting multi-stage membrane screens and 15 of which were connected in parallel with spacers in the middle to increase the filtering area and give structural strength. It could be easily expanded with the increase in food demand. The concentrated food organisms grown outdoor was proven to be unsuitable for early clam larvae as food, but it was good to the larvae larger than 600 μm and supplied as food to spat and juvenile clams.
5. Study on the intermediate culture of juvenile clams using a suspended culture system
a. Growth of juvenile clam in bottom culture and suspended culture
Clams in conventional bottom culture are exposed to radical changes of the environment at the time of ebb tide and the feeding time is limited to high tide. Alternatively, clam may grow in a submerged suspended facility which increases the feeding time and subsequent better growth. In this study, juvenile clams were raised in hanging culture facility and conventional bottom culture. As a result, shell growth and fatness of clam spats raised in the hanging culture were far superior to the clams raised in bottom culture. The parasite infection and the mortality rate were also markedly lower in the suspended culture system. Therefor, it was concluded that the hanging culture technique developed in this study can be used in intermediate culture of juvenile clams for grow out, with low mortality and faster growth.
6. Analysis of economic value of the seed production technique and industrial application
a. Plans to reduce the production cost of food organisms
It is essential to supply enough food organism to produce Manila clam seed in hatchery condition in large scale. The algae culture methods with detailed sub culture stages developed in this study can be applied in field without any special equipments. The modified media formula uses low cost agricultural fertilizer instead of expensive high grade reagents and it reduces the food organism production cost. For f/2 media needs various reagents including NaNO₃, NaH₂PO4·H₂O, Na₂SiO₃ㆍ9HO, FeCl₃ㆍ6H₂O, CuSO4ㆍ5H₂O, Na₂MoO₄ㆍ2H₂O, ZnSO₄ㆍ7H₂O, CoCl₂·6H2₂, MnCl₂ㆍ4H₂O, vitamin B12 (cyanocobalamin), Biotin, ThiamineㆍHCl, and macro-nutrients of NaNO₃ with 75 mg/L, and NaH₂PO₄ㆍH₂O with 5 mg/L, and total of 80 g is required to produce 1 ton of f/2 media. This modified media simplified reagents needed and macro-nutrients of N, P were replaced with agricultural fertilizers. The reagents used in the algae production cost less, 5~10 times less expensive than other reagents.
b. Development of efficient seed collecting methods with substrate
The efficient seed collecting method was developed for D-larvae stage, for settling larvae through umbone stage, and for transferring to the nursery ground. This method can be applied in the seed production of other marine bivalve species, which share similar growth and metamorphosis process. The technique also reduces damage or loss of the seeds during the production, and maintains good water quality. Also, production can be increased by maintaining the spats separately sorted according to the size and changing the food ratio.
Chapter 5. Results of Current Research and Future Plan for the Application
1. Study on selecting healthy broodstock for clam seed production in hatchery system
a. Evaluation of healthy broodstock clams and conditioning in indoor system
In the present study, we collected baseline information on location of a high quality broodstock clam which produces high reproductive effort, low parasite infection and high condition index. The results obtained in this study will be usefully applied in selecting quality clam broodstock for seed production in shellfish farming industry. In addition, we established the artificial spawning technique for adjusting timing of the spawning using an indoor culturing system. This technique also will be widely applied in other marine shellfish aquaculture such as oysters and ark shell.
b. Development of health measurement techniques to select healthy broodstock
To obtain good broodstock for clam seed production in hatchery condition, the clam population must have superior physiological characteristics, and a techinque to measure health condition of clam must be established. In this study we tried to develop a technique which quantify immune parameter of clams including level of DNA damage, apoptosis, necrosis and ROS production. Flow cytometry analyzed the immune parameters of clam and the technique indicated that clams from Gangjin were physiologically superior and could be a good broodstock for clam seed production. Such techniques and the research results were published in the Korean Malacological Society Journal in 2009, and a manuscript is under preparation.
c. Development of molecular marker for selecting healthy broodstock
We successfully developed the microsatellite marker derived from expressed sequence data of clam hemocyte and identified the marker polymorphism among clams in Korea. Also, these marker was found to be a useful tool to distinguish native Korean clams from oversea clam populations (Japan, China and HongKong). Accordingly, the molecular marker developed this study could be applied in identification of imported clams from abroad as well as in the study of population genetics of R. philippinarum. Genetic characterization of marine bivalves is routinely investigated using single source of DNA including 18S rDNA, ITS or COI. However, we examined the sequences of 3 nuclear genes and single mitochondrial gene to distinguish 2 clam species. The molecular technique used in this study is considered to be a powerful tool to resolve the issue of marine bivalve taxonomy. Outcome of this study is presented in international conferences, and under preparation to submit in an international journal.
2. Study on the high quality broodstock population
a. Mass production of clam seeds in hatchery condition and seed collecting technique The technique developed in this study for mass production of Manila clam seeds in hatchery could be widely used for the mass production of other marine bivalve seeds. The guideline of calm seed production in hatchery system prepared in this study could be used in the shellfish industry.
b. Mass culture of micro algae as food organism for clam seed and spats
The mass production of food organism can be utilized not only for marine bivalves seed production, but also for other shellfish seeds such as oysters, and sea cucumbers which feed on phytoplakton as food source. In this study, we used inexpensive fertilizers to reduce the production cost, scaled-up the throughput using the facilities and equipments already available at the aquaculture farms, and presented good food organisms favored by various stages of Manila clam juveniles. The mass production of food organism can be a great business opportunity with the increase in the demand of food organisms due to the restructuring of aquaculture industry.
3. Growth and parasite burden of clams raised in intertidal bottom culture and the submerged hanging culture facility
We developed a new suspended culture method for intermediate culture of juvenile clam. Juvenile clams were included in a closed net-bag and suspended underwater for grow-out. This technique was successfully applied to field condition, resulting in significantly higher growth rate and condition index with lower level of parasite infection and mortality rate compared to those of the intertidal bottom cultured clams. In particular, we developed an effective technique for reducing fouling organism, which increases the efficiency of suspended culture. Based on these achievements, we applied for a patent titled as "Fouling-reducing apparatus for bivalve suspended culture". In addition, we will also submit the remarkable result to an international journal.