초록 ▼

○ 연구결과
가.C.polykrikoides생활사 해명
(1)유해적조 코클로디니움의 휴면포자를 적조 상습해역에서 최초 발견하여 생활사 해명
:적조 발생 원인규명,지역별 적조 잠재력 및 모니터링에 활용
(2)C.polykrikoides의 일시포자(temporarycyst)를 실내 및 현장조사에서 확인하였고,영양세포의 증식과정에서 단성생식(parthenogenesis)에 의한 형성을 확인
:적조 증식 및 확산,지역별 적조 잠재력 예측에 활용
(3)Nematodinium sp.의 휴면포자 및 발아 특이성을

○ 연구결과
가.C.polykrikoides생활사 해명
(1)유해적조 코클로디니움의 휴면포자를 적조 상습해역에서 최초 발견하여 생활사 해명
:적조 발생 원인규명,지역별 적조 잠재력 및 모니터링에 활용
(2)C.polykrikoides의 일시포자(temporarycyst)를 실내 및 현장조사에서 확인하였고,영양세포의 증식과정에서 단성생식(parthenogenesis)에 의한 형성을 확인
:적조 증식 및 확산,지역별 적조 잠재력 예측에 활용
(3)Nematodinium sp.의 휴면포자 및 발아 특이성을 최초로 발견함
나.적조발생 잠재력 파악
(1)저질 중의 휴면포자의 정량법 확립
• 밀도구배원심법과 Primuline형광염색법을 조합한 와편모조류 휴면포자 정량법 확립
• 종 특이적인 primer와 probe을 이용한 Real-timePCR법을 개발하여 적용함
:적조 발생 잠재력 조사 및 현장 모니터링에 활용
(2)지역별 휴면포자 잠재력 파악
• 직접법을 통한 Nematodinium sp.의 휴면포자의 지리적 분포 및 지도 작성
• 적조발생의 잠재력인 "seedbed"로서 가능성을 입증
• Real-timePCR을 이용한 저질 중의 C.polykrikoides발생 잠재력을 확인
-지역별 분포 양상을 지도화
-2007년 이후 발생 잠재력의 급격한 감소와 적조 미발생과의 관계 확인
:지역별 적조 발생 예측 및 조기 방제에 활용
다.C.polykrikoides지역개체군의 발생/생태환경 해석
(1)지역별 C.polykrikoides의 초기 발생 차이를 확인
• 자란만(6월 초-중순)-미조(7월 상순)-욕지도(8월 상순)
(2)지역별 초기 발생의 차이는 수심과 물리적 환경요인(저층 수온 등)의 변화와 관련
(3)C.polykrikoides과 Nematodinium sp.이 거의 동시에 발생하는 특성을 확인
-코클로디니움 유해적조 초기발생의 지표종(indicatorspecies)으로 판단함
:특정 해역에서 Nematodinium의 발생을 코클로디니움 조기발생의 모니터링에 활용
라.적조생물 성장환경 및 생리특성 파악
(1)C.polykrikoides의 최대성장속도는 수온 25℃,염분 30psu에서 0.35day-1로서
염분의 증·감에 따라 성장속도가 급격히 변화하는 것을 확인
:현장의 관측(자란만 집중조사)결과와 잘 일치
(2)C.polykrikoides는 DIP가 제한된 환경에서 DOP 이용 가능성을 확인
:유기물 유입이 많은 양식장 인근의 유해적조발생 모니터링 및 방제에 활용
(3)유해 와편모조류 Prorocentrum minimum과 Heterocapsapygmaea의 종특이적
살조 바이러스를 발견,숙주세포를 사멸시키는 특성을 입증(특허 출원)
:C.polykrikoides을 포함한 유해적조생물 제어에 활용 가능성
마.적조 발생 확산 및 예측모델 개발
(1)남해 동부해역(자란만-욕지도 라인)에서 해양물리조사를 통한 유동모델 구축
(2)해수 유동모델과 Lagrangian방법을 이용한 입자 추적자 모델을 확립
:유해적조(HABs)의 거동(확산)파악,적조예찰 및 방제에 활용
바.생활사 단계에 따른 적조 제어기술 개발
(1)적조 잠재력(seedpotential)제어 현장 평가 및 초기 발아개체군 구제 가능성 검토
-Real-timePCR을 이용 C.polykrikoides영양세포의 구제 효율 확인(실내실험)
-조사기간 동안 적조 미발생으로 현장조사 불가
:적조 구제물질 살포에 대한 효과 및 잠재력 제어 효율성 검증에 활용 기대됨

Abstract ▼

IV. Results
1. Life cycle elucidation of Cochlodinium polykrikoides
(1) The first findings of resting cysts of Cochlodinium polykrikoides and Nematodinium sp. and the identification of their germination
To identify the reality of resting cysts of C.polykrikoides, we conducted a cultivation

IV. Results
1. Life cycle elucidation of Cochlodinium polykrikoides
(1) The first findings of resting cysts of Cochlodinium polykrikoides and Nematodinium sp. and the identification of their germination
To identify the reality of resting cysts of C.polykrikoides, we conducted a cultivation and microscope examination on suspended materials sieved in filtered seawater after sampling sediments from Saryang-do and Jaran Bay, where there were annually reported periodic formations of harmful algal blooms and early out breaks of vegetative cells. We conducted a net sampling(20 ㎛) of vegetative cells, and observed floating cysts at the same time.
Even though no germination was traced directly in cysts of C.polykrikoides from the sediment under the sampling process, the occurrence of its vegetative cells, germinants, was verified. On the other hand, the resting cysts of C.polykrikoides were first found in the net sampling at the periodic examination on the station 4 in Jaran Bay from the end of May through early June, 2009 and their gemination process was verified under the microscope. Just before germination, resting cysts were observed to be with a small spine and without one, and all of them had a conspicuous red body.
Theirsize was 35～43 ㎛ long, and they were distinguished from hyaline cysts being formed in the process of germination. The early germinants were 35∼45 ㎛ long and 20∼30 ㎛ wide, and their vegetative cells developed into 8 chains at the maximum.
(2) The formation of hyaline cysts and characteristics of their germination
Hyaline cysts formed from C.polykrikoides were similar to vegetative cells in size, pale in color, immobile without chloroplasts. Their size was similar to that of motile cells. Only faint traces of the sulcus and cingulum remained on the surface ; all hyaline cysts were surrounded by a transparent, thin hyaline membrane. After being preserved for 6 months at 4ºC in darkness, cells of C.polykrikoides regenerated successfully from the hyaline cysts when moved to the light and a higher temperature. Hyaline cells developed diversely into single cells or cell clusters being chained with eight cells. Each cell had a red body, and cell clusters formed a hyaline membrane surrounding cells. Even in the sediment, cells could be detected having primuline staining. Thanks to their chloroplasts emitting red light, they were determined to be alive under the fluorescence microscope. The result meant that most vegetative cells of cell clusters chained with more than four cells could be transformed in to hyaline cells without changing the number of cell clusters. It also suggested that hyaline cells could be considered a kind of temporary cysts, not to be at the dying stage of vegetative cells. As C. polykrikoides cells regenerated vegetative cells successfully from the hyaline cysts in the favorable condition, hyaline cells were determined to be in one stage of the life cycle of C.polykrikoides.
(3) The occurrence and systematic interpretation of Nematodinium sp. as an indicator species of C. polykrikoides
C. polykrikoides and Nematodinium sp. were observed to share some similarity in the outbreaking period and in the morphology of germinants.
Resting cysts of Nematodinium sp. were present all over the area of the South Sea, Korea. As C. polykrikoides and Nematodinium sp. began to occur on the spot at the same time and showed a similar aspect of occurrence, the two species were estimated to play as a indicator species to each other in the mode of ecological occurrence. In order to grasp an accurate systematic relationship of Nematodinium sp., an interpretation of molecular system was conducted by using the information of genome sequence of dinoflagellates registered in NCBI. Nematodinium sp. in the experimental group was interpreted to be divided from groups of Prorocentrum and Lepidodinium and from one clade. There was no species being perfectly coincident with Nematodinium sp. in the system analysis. The most related species were Warnowiasp. and Nematodinium sp., being followed by Cochlodinium cf. geminatum and Dissodinium. They were much similar in morphology, but as they showed a partial genetic difference in the result of this study, further detailed morphological and molecular biological researches were required for a definite classification of species.
2. The development and application of conventional method for the discrimination of resting cyst potentials of C. polykrikoides
(1) The establishment of the counting method of resting cyst
The method of SPT was introduced to count resting cysts directly in the sediment. The resting cysts of dinoflagellates were reported to have a specific gravity of about 1.3 g/cm³(Anderson and Lively, 1985; Bolch, 1997).
The most dinoflagellates could be separated from an experimental group with a concentration of 1.3 g/cm³, and they were more than from experimental groups with a concentration of 1.2g/cm³ and 1.5 g/cm³.
Afterwards, the concentration of SPT solution was set at 1.3 g/cm³ for resting cysts of Nematodinium sp. in the quantitative analysis.
Vegetative cells of C. polykrikoides were morphologically defined by their distinct characteristics of the cingulum, and were identified by light microscopy and/or scanning electron microscopy (SEM) on cultured strains(Matsuoka et al.2008). For identification of dinoflagellate cysts, light microscopy and SEM observations were also required, and the field-derived cysts were commonly incubated in culture media over a period of several weeks for growth, and then the germinated cells were re-examined by microscopy for species verification. These conventional methods required considerable time and expertise for species identification. To overcome this difficulty, molecular identification methods such as real-time PCR assay, fluorescent in situ hybridization and sandwich hybridization have been used for detection of harmful dinoflagellates. The rapid and accurate identification and enumeration of the target species of interest were prerequisites for HABs monitoring programs. Real-time PCR assay has been used for rapid detection and enumeration of harmful dinoflagellates in waters and sediments. The method used a cloned fragment of the internal transcribed spacer 2(ITS2) rDNA gene as a standard for cyst quantification.
(2) The meaning of the distribution investigation of resting cysts by the direct counting method
A common assumption was that cyst ‘seed beds’ provided the inoculum for many harmful blooms. Given the widespread cyst distribution typical of coastal areas, one important scenario was that the blooms were initiated by the synchronized germination of cysts throughout the region. Therefore, we investigated the cyst distribution along the coastal areas such as Gosung-Jaran Bay and Wando, where Cochlodinium blooms were prevalent in 2007. As expected, a high distribution of Nematodinium cysts was shown only in some stations of the coastal areas where blooms occurred last year(10-15 cysts/10 g sediments in wet weight) and in stations where a physical accumulation was possible(98 cysts/10 g sediments), while the other stations showed just a trace of their distribution(0-2cysts/10 g sediments).
On the other hand, from their feature that the cyst population germinates under favorable environmental conditions, it was estimated that their densely-populated regions took the role of point sources of recurrent bloom initiation in the shallow coastal areas.
(3) An investigation of regional cyst potential by using real-time PCR method
C. polyrkikoides was detected in sediment samples from the coast of Southern Sea. Of total sediment samples, Jaran Bay samples were positive for C. polykrikoides continually(∼2010), other location samples were detected.
The abundances of these species in the coast of Jaran Bay were relatively higher than other location(100r DNA copies for C. polykrikoides per cm³ of wet sediment obtained from top layer of centrifuged 30 g of sediment). C. polykrikoides was detected from the coasts of Saryangdo, Yeosu, Wando, Mijo-myeon, Tongyeong,Yokji-do from 10 to 30 rDNA copies per cm³ of wet sediment. As a result, the detection of resting cysts of C. polykrikoides from samples in the sediment by real-time PCR can be used to clarify the potential of red tide breakout and to predict the occurrence of resting cysts of C. polykrikoides in the water mass. It can be used in locating regions liable to the red tide outbreak and in establishing a device to minimize the harm of the red tide.
3. An Interpretation on the occurrence and the ecological environment of a regional population of C. polykrikoides
(1) An periodic investigation on strains of Jaran Bay, Yokji-do, Saryang-do and Mijo-myeon
Physico-chemical and biological factors were investigated on seawater and sediment samples in the areas of Jaran Bay, Yokji-do, Mijo-myeon, and Saryang-do, that is, major regions where C. polykrikoides occurred from 2009 through 2011.
Around Jaran Bay from May through September, water temperature was 16.88∼31.05℃ at the surface and 14.65∼25.90℃ at the bottom, salinity was 20.35∼34.94 psu at the surface and 30.10∼35.02 psu at the bottom, and dissolved oxygen was 4.00∼10.76㎎/L at the surface and 0.79∼8.10 ㎎ / L at the bottom. DIN(dissolved inorganic nitrogen) was in the range of 0.08∼2.68 μM at the surface and 0.03∼7.84 μM at the bottom, and DON(dissolved organic nitrogen) was 0.21∼2.80μM at the surface and 0.05∼1.64 μM at the bottom. DIN and DON accounted for fifty percent of DTN(dissolved total nitrogen) at the surface, respectively and DIN accounted for sixty five percent of DTN at the bottom. DIP(dissolved inorganic phosphorus) was 0.09∼1.62 μM at surface and 0.02∼1.81 μM at the bottom, and DOP(dissolved organic phosphorus) was in the range of 0.03∼0.56 μM at the surface and in the range of 0.03∼0.52 μM at the bottom. DIP accounted for seventy percent of DTP(dissolved total phosphorus) at the surface and for sixty five percent at the bottom. DSi(dissolved silicon) was in the range of0.13∼13.51 μM at the surface and in the range of 1.15∼12.98 μM at the bottom.
As for the species composition and standing crops of phytoplankton, 35 species of dinoflagellates (300∼27,000 cells/L) and 36 species(2,100∼2,000,000 cells/L) were observed among 71 species of phytoplankton, and their standing crops increased with the lapse of time from spring to summer. The diatom Chaetoceros sp.was a dominant species in the period of investigation. C. polykrikoides tended to increase correspondingly with the increase of diatoms at the early observation, but did not develop to the point of blooms under the influence of dominant diatoms.
In Saryang-do from July through August of 2010 and 2011, water temperature was in the range of 23.56∼25.24℃ at the surface and 18.85∼20.51℃ at the bottom. Salinity was 31.58∼34.23 psu at the surface and 32.21∼34.34 psu at the bottom. DO was in the range of 6.73∼7.49㎎/L at the surface and in the range of 4.18∼6.51㎎ / L. As for the species composition and standing crops, 14 species of dinoflagellates(400∼3, 400 cells/L) and 28 species of diatoms (8,000∼459,350cells/L) were observed among 42 species of phytoplankton. The diatom Pseudo-nitzschia pungens was dominant species.
Germinants of C. polykrikoides were observed to be with a concentration of 45∼155cells/L at the investigation on July 21, and did not develop into a mass occurrence.
In Mijo-myeon in July and August, 2011, water temperature was 22.73∼25.80℃ at the surface and 17.29∼24.41℃ at the bottom, salinity was 27.79∼33.44 psu at the surface and 32.38∼34.93 psu at the bottom, and dissolved oxygen was 4.52～6.86㎎/L at the surface and 1.45～5.16㎎/L at the bottom. DIN was in the range of 0.87∼1.97μM at the surface and in the range of 0.85∼1.95 μM at the bottom. DON was in the range of 0.04∼0.66 μM at the surface and in the range of 0.12∼1.59μM at the bottom. DON accounted for twenty percent of DTN at the surface and for about 30 percent of DTN. DIP was in the range of 0.01∼0.53 μM at the surface and in the range of 0.06∼0.69μM at the bottom, and DOP was 0.03∼0.38 μM at the surface and 0.08∼0.38 μM at the bottom. DOP accounted for fifty percent of DTP. DSi was in the range of 0.54∼10.63 μM at the surface and in the range of 1.35∼9.24 μM at the bottom.
Among 52 species of phytoplankton, 22 species of dinoflagellates marked a difference in standing crops of 310∼6,500 cells/L, and 30 species of diatoms were observed in standing crops of 67,050∼100,400 cells/L. After the diatom Asterionella glacialis became dominant, there was species succession in to Chaetoceros pseudocurvisetus and Skeletonema costatum. Germinants of C. polykrikoides were found to beat 20∼200 cells/L on July 26, and at 5∼42 cells/L on August 24.
In Yokji-do from July through August, 2011, water temperature was 22.73∼25.80℃ at the surface and 17.29∼24.41℃ at the bottom. Salinity was 27.79∼33.44 psu at the surface and 32.38∼34.93 psu at the bottom, and dissoved oxygen was 4.52～6.86㎎/L at the surface and 1.45～5.16㎎/L at the bottom. DIN was 0.31∼1.98 μM at the surface and 0.824∼1.97 μM at the bottom, and DON was 0.04∼0.66 μM at the surface and 0.12∼0.74 μM at the bottom, accounting for thirty percent and thirty five percent of DTN, respectively. DIP was in the range of 0.00∼0.32μM at the surface and in the range of 0.01∼0.38 μM at the bottom, and DOP was 0.03∼0.44 μM at the surface and 0.03∼0.38 μM at the bottom, accounting for fifty percent and forty percent of DTP, respectively.
12 species of dinoflagellates(1,500∼10,000cells/L) and 27 species of diatoms(21,900∼613,200cells/L) were observed among 39 species of phytoplankton. Just like in Mijo-myeon, A. glacialis was dominant, being followed by C. pseudocurvisetus and S.costatum. Some germinants of C. polykrikoides were traced on July 26 and August 24, but did not develop in to ared tide out break.
(2) A concentrative investigation on fixed stations in Jaran Bay
During the concentrative investigation, germinants started to be detected in June, 2010 and 2011, distributing evenly in the seawater on the surface and at the bottom. Mostly, they occurred first at the bottom, and later the number of germinants tended to increase at the surface. This change can be understood as a response of germinants to the change of conditions in diverse environments such as water temperature. In 2011, there was no detection of resting cysts at the bottom, but Jaran Bay could be determined to be a habitual region of C. polykrikoides occurrence, as its germinants were detected in these a water.
(3) A correlation in the occurrences of germinants of Cochlodinium polykrikoides and Nematodinium sp.
Through periodic and concentrative investigation, germinants of C. polykrikoides and Nematodinium sp. were found to appear almost at the same time. In the investigation of the occurrence of germinants of C. polykrikoides and Nematodinium sp. from May 29 through August 17, 2010, Nematodinium sp. appeared to be with a concentration of 14 cells/L and 7 cells/ L at the station 4 and 7 on June 13, respectively, and was followed by the occurrence of C. polykrikoides on June 16. At tha ttime, Nematodinium sp. showed a high concentration of 45 cells/L at the station 4 and of 54 cells/L at the station
7. In the investigation from May 14 through August 3, 2011, the occurrence of germinants of C. polykrikoides and Nematodinium sp. was found to show a tendency similar to that in 2010, and germinants of C. polykrikoides were detected two or three days after the discovery of germinants of Nematodinium sp. As Nematodinium sp. and C. polykrikoides occurred on the spot almost at the same time, showing a tendency of occurrence similar to each other, they can be considered to play a role of an indicator species of initial development to each other, and they can be used in monitoring the initial development of C. polykrikoides in some specific sea area on the basis of the consideration.
4. A detection of growth conditions, and growth promoting or inhibition components for harmful algae
(1) An Identification of growth environment and physiological characteristics of Cochlodinium polykrikoides
To understand the physiological characteristics of harmful dinoflagellate C. polykrikoides, we investigated the effects of water temperature, salinity, light and nutrients on its grow th, using strains isolated from the coastal area of Korea.
As for the effect of water temperature and salinity on the grow th of C. polykrikoides, the cell growth was not observed at a salinity of 15 psu and 20 psu at a temperature of 15℃, but other experimental groups grew. The maximum growth rate(0.35day^-1) was obtained at 25℃ and at a salinity of 30 psu. The growth rate of C. polykrikoides depended conspicuously on the change of salinity rather than on that of water temperature, and the optimum salinity of C. polykrikoides was a little higher than that of other red tide species in Korea. In the light experiment, C. polykrikoides did not grow at 10 μmol m^-2s^-1 and its cell growth was observed at irradiance values of 25 μmol m^-2s^-1 and above. The irradiance-growth curve was described as μ=0.30(I-15.27)/(I+27.22),(r=0.99). This suggests a compensation PFD of 15.27 μmol m^-2s^-1 and a maximum growth rate of 0.30 day^-1. In conclusion, C.polykrikoides preferred a high salinity, temperature and irradiance in the summer of Korea. This result provided important information for understanding the mechanism of C. polykrikoides blooms and for developing the technology to predict the blooms of this organism in the field.
To understand the affinity of C. polykrikoides to nutrients, we experimented on its uptake kinetics of DIP(dissolved inorganic phosphorus), one of typical limiting nutrients in the coastal area. Its uptake rate increased correspondingly on the increase in the concentration of DIP up to 10 μM, but showed no more difference at a higherconcentration than10μM. The
Michaelis-Menten's equation showed a high determination coefficient, r=0.90. At that time, the maximum uptake rate(ρmax) was 0.80p molcell^-1hr^-1 and half-saturation concentration(Ks) for DIP was 1.87 μM. In the present study, the Ks value of C. polykrikoides was lower than that of Gymnodinium catenatum and Alexandrium tamarense, but it was higher than that of the diatom Skeletonema costatum. This result suggested that C. polykrikoides was disadvantaged in competition with the diatom Skeletonema costatumin an environment of inorganic nutrients. Phytoplankton with a high ρmax could survive by storing nutrients in the cell even though the high Ks was quite disadvantaged in competition. The maximum specific uptake rate (Vmax; ρmax/Q₀) of C.polykrikoides was estimated to be 51.9 day^-1. This value was high, compared to those reported on other species. The Vmax/Ks was calculated to be 27.7, which was similar to that of A. tamarense. This indicated that C. polykrikoides was a poor competitor in terms of utilizing inorganic nutrients, compared to the dominant diatom in the coastal area of Korea.
C. polykrikoides maintained its growth utilizing DOP(dissolved inorganic phosphorus) compounds, which had as various molecular weights and structures as DIP. The growth rate of PME(phosphomonester) was observed to be 0.13～0.19 day^-1, which was correspondent to 92±12% in comparison with that of Ortho-P(orthophosphate). As for DOP compounds in the nucleotide such as AMP(adenosine 5 -monophosphate), ADP(adenosine 5-diphosphate) and ATP(adenosine 5 - triphosphate), their growth rate was similar to that of Ortho-P. Thus, C.polykrikoides might grow, utilizing DOP compounds under DIP-limited conditions. In other word, the DIP-limited condition would give a favorable influence on the growth of C.polykrikoides.
In Alkaline phosphatase(APase) activity, which hydrolyzes phosphomonoesters in the surrounding water, the APase of C.polykrikoides increased with its cell growth, while DIP concentration decreased in the culture fluid. Its APase reached a maximum of activity at the stationary phase of cell growth. The APase activity of C.polykrikoides was first detected when the DIP concentration decreased to 0.83 μM(day 11). The APase reached a maximum of 0.70 pmol cell^-1hr^-1 in the stationary phase. DIP concentration with the first induction of the APase activity was considerably higher than that in Jaran Bay. Therefore, organic nutrients suchas DOP may be useful for the survival, cell division and species competition of C. polykrikoides.
(2)Separation and physiology and ecological characteristics of algicidal viruses
Most of viral particles were presumably bacteriophages infecting marine germs, but viruses infecting algae have also been reported. In this study, a virus screening was done by the microplate MPN(The Most Probable Number) method on 0.22 ㎛filtered seawater samples from each sampling site. As a result, Heterocapsa pygmaea infecting virus (HpygDNAV01) was detected to infect and lyse the dinoflagellate Heterocapsa pygmaea and Prorocentrum minimum. A further discovery of viruses infecting other harmful algae including C. polykrikoides would make it possible to restrain harmful algal blooms on the basis of this study.
5. The establishment of a model for mitigation and prediction of harmful algal blooms
(1) An physical investigation and the establishment of a hydrodynamic model
Tidal currents flowed to northwest at the maximum flood in the spring tide, and to southeast at the maximum ebb in the spring tide. Neap tidal currents showed a similar distribution in the maximum flood and the maximum ebb to that in the spring tide, but the speed decreased compared to that in the spring tide. As for the distribution of the tide-induced residual current, the flow to southeast was found to be mostly prevalent.
(2) Particle Tracking Model
The movement of harmful algal blooms was detected with the particle tracking model using a hydrodynamic model and Lagrangian method.
Currents with four tidal components, that is, M2, S2, K1,O1, were calculated with the help of POM(Princeton Ocean Model) in the coastal area of Tongyeong and Namhae. A particle tracking model was utilized to understand the spatial distribution of harmful algal blooms with the lapse of time in the case of their occurring in Jaran Bay, the sea off Mijo-myeon and the west of Yokji-do, and also in the case of the simultaneous occurrence at the three areas.
Harmful algal blooms in Jaran Bay could distribute 78 percent of harmful algal particles two days after their occurrence, and spread to the southern water of Namhae and Yokji-do with the lapse of four days. With the lapse of six days, they spread to a faraway sea of Yokji-do and turned to east slowly. In the case of harmful algal blooms in the sea off Mijo-myeon, they distributed mostly in the south and the west of Namhae-do until the lapse of four days, but they spread to the outer waters largely five days later.
Eight days later, they distributed largely in the south water of Namhae-do again and proceeded to north through the west channel of Namhae-do. Ten days later, harmful bloom particles flew in to Jinhae Bay after proceeding to north through the west channel of Namhae-do.
6. An evaluation of field restraint on HABs seed potential and examination about the possibility of mitigating the initial germinant population
(1) An investigation of the mitigation efficiency of loess spreading on vegetative cells of Cochlodinium polykrikoides
As there was no outbreak of C. polykrikoides blooms on the spot during the research, the mitigation efficiency of loess spreading was investigated in the indoor experiment. With the vegetative cells of C. polykrikoides being with a concentration of 1,000 cells/mL, 2,000 cells/mL and 5,000 cells/mL, and loess being spread in the range of 0～10,000 ppm, the chlorophyll a of experimental groups were measured one day after the spread of loess, being compared to that of control groups. As a result, the mitigation efficiency was the highest on vegetative cells of C. polykrikoides with a concentration of 2,000 cells/mL and 10,000 ppm of loess. The efficiency should be identified in mass culture condition with the installation of mesocosm on the basis of this experiment.
(2) An investigation of the effect of loess spreading in microcosm experiments
Microcosm experiments were done on sterilized sediment and f/2 culture medium in cell culture flask in order to investigate the ability of restraining the formation of resting cysts. Loess was spread with a concentration of 10,000 ppm over C. polykrikoides cultured at 5,000 cells/mL, and real-time PCR was adopted to detect vegetative cells and resting cysts. As a result, C. polykrikoides was not detected in the sediment before the spread of loess.
One hour after the spread of loess, the value of C. polykrikoides were similar at the surface and bottom. However, the value of C. polykrikoides was found to increase in the middle of water, and to tend to decrease at the bottom. It was found that the cells did not undergo a cell destruction and C. polykrikoides having the ability of swimming rose from the bottom to the surface, while the spread of loess was settling a physical coagulation of vegetative cells at the bottom. C.polykrikoides being detected in the sediment ten days later were determined to be temporary cysts. Therefore, a long- term field investigation was expected to be adopted on the field of the red tide potential using real-time PCR.