Studies on mutual interaction of biological factors and blooming dynamics in Cochlodinium polykrikoides (Dinophyceae)

Abstract

It has been recently known that biological factors play a significant role in the bloom dynamics of harmful algae. However, biological factors have received relatively little attention compared to physiochemical factors. Thus, studies on biological factors are necessary to better understand bloom mechanisms. The bloom mechanisms of toxic dinoflagellate Cochlodinium polykrikoides which was mainly focused on physiochemical factors has not been fully understood yet. Accordingly, this study aimed to improve the understanding of bloom mechanisms through analyzing mutual interaction between C. polykrikoides blooms and biological factors, such as genetic differentiation within C. polykrikoides, bacterial community structures and endoparasite Amoebophrya species. The toxic dinoflagellate C. polykrikoides forms blooms that are maintained for longer periods (≥8 weeks) than other red tide species in Korean coastal waters and has remained as a mystery. Although the genetic differentiation would be the one of possible explanation of this mystery, there was no tool for investigating the impact of genetic differentiation. In this study, to understand this, a real-time PCR assay to identify and quantify C. polykrikoides at the intraspecific level was developed. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r2≥0.995) were established between Ct and the log of the cell number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms were found to be comprised of Philippines and East Asian ribotypes, but not the American/Malaysian ribotype in Korean coastal waters. The Philippines ribotype was found to be highly abundant during summer (≥23oC) peak, whereas the East Asian ribotype became the dominant ribotype as the bloom continued on fall (≤19oC) in 2009. Even though these ribotypes belong to the same species, they may have completely different ecological traits induced by genetic differences. Duration of C. polykrikoides blooming in 2009 was much longer than that in 2010. Interestingly, in 2010, the only one ribotype was responsible for C. polykrikoides blooming, whereas ecological succession between the two ribotypes of C. polykrikoides occurred in 2009. East Asian ribotype was detected near southern Korean coastal waters, whereas Philippines ribotype was distributed near Ieodo and Cheju where directly affected by Kuroshio Currents. Hence, Philippines ribotype may have been originated from outer area via Kuroshio Currents. To investigate bacterial community structures followed by different C. polykrikoides bloom stages (before-, early-, middle-, late-bloom and bloom termination), cloning library analysis was used for this study since it allows the analysis of a detailed phylogenetic diversity and composition proportion of bacterial communities. In this study, α- and γ-proteobacteria were thought to be important phyletic groups during C. polykrikoides blooms in Korean coastal waters. The proportion of γ-proteobacteria which was known as the algicidal group was lowered (11.8%) at the middle of blooming phase. However, since then, Gammaproteobacteria comprised of high proportion (26.2%) when C. polykrikoides bloom was vanished. Whereas, Alphaproteobacteria showed a clear dominance during the bloom period and primary member of Rhodobacterales were abruptly increased from 37.5% (before bloom) to 73.7% (early bloom). High proportion of Rhodobacterales was maintained until late bloom stage (56%). Moreover, site Jinmok (50%) and Bangjuk-po (35.3%) where C. polykrikoides bloom occurred in October 2012 also contained high portion of Rhodobacterales. In principal component analysis (PCA), Rhodobacterales had a statistically significant relationship with C. polykrikoides bloom (P-value ≤0.001). Since, there is no ecological study about Amoebophrya sp. killing C. polykrikoides. Dinospore which is free-living form of Amoebophrya is directly related with infection rate of host cell. Although dinospore may suspected to be important to C. polykrikoides bloom termination, there is no tool to identify and quantify dinospore accurately due to its extremely small body-size (<5 μm). To investigate impacts of Amoebophrya spp. on C. polykrikoides bloom termination, qPCR assay based on copy number was developed. As results, highly significant standard curves (r2≥0.99) and one informative melting curves showed the accuracy of this quantitative assay. During this study, C. polykrikoides blooms occurred twice at site of Mulgun Harbor, in August and October of 2012. Using qPCR assay, the free-living cells showed similar dynamics to those during their parasitic stage living inside host cells. While Amoebophrya sp. 1 was predominant in August, Amoebophrya sp. 2 was strongly related with the termination of the C. polykrikoides bloom in October. Moreover, daily induced mortality reached a maximum (29.55%) a week before the C. polykrikoides bloom abruptly vanished. Accordingly, these two Amoebophrya species are thought to play an important role in terminating C. polykrikoides blooms in southern Korean coastal waters. Previous studies regarding C. polykrikoides bloom mechanisms were mainly focused on physiochemical factors, but they could not fully clarify bloom mechanisms of C. polykrikoides. Results of the present study suggested new insights of bloom mechanisms of C. polykrikoides. Ecological succession between the two ribotypes of C. polykrikoides may contribute on long bloom periods. Bacterial communities strongly interacted with C. polykrikoides blooms and Rhodobacterales may play an important role in bloom initiation. Finally, endoparasite Amoebophrya spp. was thought to be a direct trigger to mitigate C. polykrikoides bloom in Korean coastal waters.|Cochlodinium polykrikoides 적조 발생 및 소멸 기작 규명을 위해, C. polykrikoides의 개체군내의 리보타입(ribotype)에 대한 생태적 거동, 박테리아 군집 및 기생성 와편모류 Amoebophrya의 C. polykrikoides 생태적 거동에 미치는 영향을 연구하였다. 기존 연구에서 유전적인 차이로 인한 C. polykrikoides의 sub-population인 3개의 리보타입이 존재한다는 것이 밝혀졌음에도 불구하고, 방법론적인 한계로, 이들 리보타입에 대한 생태적 거동연구는 전무한 실정이다. 따라서, 본 연구에서는 C. polykrikoides내의 세가지 리보타입에 대한 정량 및 정성 분석이 가능한 리얼타임(real-time) PCR기법을 개발하여 이들 리보타입의 생태적 거동을 연구하고자 하였다. 이를위해, C. polykrikoides의 세가지 리보타입에 대한 특이적인 분자탐침을 28S 리보조말(ribosomal) RNA를 기반으로 제작하였다. 또한, 멜팅커브(melting curve)분석으로 특이적인 분자 탐침이 대상 리보타입만을 증폭하는 것을 확인하였으며, 정량분석을 위한 검량선은 매우 높은 유의성(r2≥0.995)을 보였으며, 리얼타임 PCR 기법을 통해 정성 및 정량분석이 가능함을 확인하였다. 리얼타임 PCR 기법을 현장적용한 결과, 미국/말레이시아 리보타입을 제외한 필리핀, 동아시아 리보타입이 한국 연안에서 분포함이 밝혀졌다. 2009년과 2010년에 C. polykrikoides 적조가 발생한 현장시료를 대상으로 리얼타임 PCR기법을 이용하여 리보타입의 생태적 거동을 조사하였다. 그 결과, 비교적 긴 기간동안 적조가 발생했던 2009년의 경우 필리핀과 동아시아 리보타입이 연속적으로 천이되어 C. polykrikoides 적조가 발생한 반면, 2010년의 경우 동아시아 리보타입만이 적조를 유발하였다. 흥미롭게도, 두개의 리보타입은 서로 다른 생태적 특성이 관찰되었다. 2009년의 모든 조사정점에서, 필리핀 리보타입은 수온이 높은 여름(≥23oC)에, 동아시아 리보타입은 수온이 비교적 낮은 가을(≤19oC)에 우점하였다. 따라서, C. polykrikoides 적조의 장기화현상은 생태적으로 다른 특성을 가진 리보타입이 연속적으로 천이되면서 발생하는 것으로 판단된다. 동아시아 리보타입은 한국 남해 연안 주변과 비교적 거리가 가까운 제주도 일부 정점에서만 탐지된 반면, 필리핀 리보타입은 제주도 주변 해역뿐 아니라, 이어도 주변해역까지도 높은 밀도(19,698±843 cells liter-1)로 분포하였다. 이는 필리핀 리보타입이 쿠로시오 해류를 통해 외부에서 유입되었을 가능성을 시사한다. 적조 발생기작으로 물리-화학적인 요인과 생물학적 요인과의 상호작용에 대한 연구는 비교적 많이 수행되어 왔음에도 불구하고, C. polykrikoides 적조의 발생과 소멸을 설명하기에는 많은 한계점을 갖고 있다. 최근 박테리아 군집은 와편모조류 적조 발생과 매우 밀접하다고 밝혀졌다. 그러나 C. polykrikoides 적조와 박테리아 군집과의 연구는 전무한 실정이다. 본 연구에서, 박테리아의 군집과 적조 발생간의 상관관계를 규명하기 위해, cloning library 기법을 사용하여, 2012년 적조가 발생했던 물건항에서 적조 발생전, 발생 초기, 중기, 말기, 소멸시기를 구분하여 박테리아의 군집구조의 변화를 분석하였다. 그 결과, 살조효과를 나타낸다고 알려진 Gamma-proteobacteria는 발생 중기시 낮은 비율(11.8%)로 나타냈으며, 적조 소멸이후 26.2%로 유의성있게 증가하였다. 반면, Alphaproteobacteria는 적조발생 초기에 가장 높은 비율을 나타냈으며(34.5%), 이 중 Rhodobacterales의 비율은 적조발생전과 비교할 때, 37.5% 에서 발생초기에 73.7%로 유의적으로 증가하였을 뿐 아니라, 발생 중기(48.6%) 및 말기(56%)까지도 지속적으로 높은 비율을 유지하였다. 또한, C. polykrikoides 적조가 발생했던 진목(50%)과 방죽포(35.3%)에서도 Rhodobacterales가 높은 비율을 차지했다. 이러한 결과들의 유의성을 조사하기 위해 주성분 분석 결과, C. polykrikoides 적조와 Alaphaproteobacteria에 속하는 Rhodobacterales는 통계적으로도 매우 밀접한 관계를 가지고 있음을 알 수 있었다(P-value ≤0.001). Talyor (1968)에 의해 최초로 기생생물이 적조소멸에 중요한 역할을 한다는 것이 알려진 이후, 와편모조류 적조소멸과 기생 와편모류 Amoebophrya의 관계에 대한 다수의 연구가 진행되어 왔으나, C. polykrikoides의 적조소멸과 Amoebophrya 관계 연구는 전무하다. 이는 Amoebophrya의 경우, 자유유영 세포인 다이노스포어(dinospore)는 감염률과 직접적으로 연관되기 때문에 적조소멸과 밀접한 관계를 가지고 있는 다이노스포어 생태적 거동연구는 매우 중요하다. 그러나, 이들의 작은 세포크기(≤5 μm)때문에, 현장에서 광학현미경으만으로 다이노스포어의 탐지 및 정량분석은 한계가 존재한다. 이러한 방법론적인 한계점을 극복하기위해, 이들 기생와편모류의 ITS 영역을 대상으로 특이적인 분자탐침을 제작하고, 유의성이 높은 검량선(r2≥0.99)과 멜팅커브 분석을 통해 정량적 리얼타임 PCR기법을 개발하여 적조 소멸과 Amoebophrya의 연관성을 연구하였다. 물건항에서 8월과 10월에 C. polykrikoides 적조가 발생하였으며, 정량적 리얼타임 PCR기법을 사용한 결과, Amoebophrya sp. 1은 8월에, 그리고 Amoebophrya sp. 2는 10월에 숙주세포인 C. polykrikoides 적조가 발생하였을때, Amoebophrya sp. 1과 2의 다이노스포어 및 감염단계의 카피(copy)수가 유의적으로 증가하였으며, Amoebophphrya 증가 이후, 숙주세포인 C. polykrikoides의 세포수는 급감하였다. 특히, Amoebophrya sp. 2의 경우, 카피수가 최대값을 나타냈을 때(10월), 숙주세포에 대해, 높은 감염률(45%)을 나타냈으며, 이로 인한 숙주세포의 사망률은 29.55%로 Amoebophrya는 한국연안에서 C. polykrikoides 적조 소멸에 중요한 역할을 수행하는 것으로 판단된다. 그동안 한국 연안에서는 C. polykrikoides가 물리, 화학적 요인조건과 관계없이 적조 발생 및 소멸현상이 나타나, C. polykrikoides 적조 기작 연구에 많은 어려움이 있었다. 그러나, 본 연구를 통해 한국연안의 C. polykrikoides 적조 대발생 기작에 대한 새로운 시각을 제공할 수 있었다. 먼저, 두개의 리보타입이 연속적으로 천이되어 C. polykrikoides 적조 장기화의 발생한다는 사실을 밝혔으며, 박테리아 군집은 C. polykrikoides 적조와 매우 밀접한 관계를 가지며, 특히, 적조 발생하는데 있어서 Alphaproteobacteria에 속하는 Rhodobacterales는 중요한 역할을 수행하는 것으로 나타났다. 마지막으로, 기생 와편모류인 Amoebophrya 는 한국연안의 C. polykrikoides 적조 소멸의 주요 요인으로 사료된다.; It has been recently known that biological factors play a significant role in the bloom dynamics of harmful algae. However, biological factors have received relatively little attention compared to physiochemical factors. Thus, studies on biological factors are necessary to better understand bloom mechanisms. The bloom mechanisms of toxic dinoflagellate Cochlodinium polykrikoides which was mainly focused on physiochemical factors has not been fully understood yet. Accordingly, this study aimed to improve the understanding of bloom mechanisms through analyzing mutual interaction between C. polykrikoides blooms and biological factors, such as genetic differentiation within C. polykrikoides, bacterial community structures and endoparasite Amoebophrya species. The toxic dinoflagellate C. polykrikoides forms blooms that are maintained for longer periods (≥8 weeks) than other red tide species in Korean coastal waters and has remained as a mystery. Although the genetic differentiation would be the one of possible explanation of this mystery, there was no tool for investigating the impact of genetic differentiation. In this study, to understand this, a real-time PCR assay to identify and quantify C. polykrikoides at the intraspecific level was developed. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r2≥0.995) were established between Ct and the log of the cell number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms were found to be comprised of Philippines and East Asian ribotypes, but not the American/Malaysian ribotype in Korean coastal waters. The Philippines ribotype was found to be highly abundant during summer (≥23oC) peak, whereas the East Asian ribotype became the dominant ribotype as the bloom continued on fall (≤19oC) in 2009. Even though these ribotypes belong to the same species, they may have completely different ecological traits induced by genetic differences. Duration of C. polykrikoides blooming in 2009 was much longer than that in 2010. Interestingly, in 2010, the only one ribotype was responsible for C. polykrikoides blooming, whereas ecological succession between the two ribotypes of C. polykrikoides occurred in 2009. East Asian ribotype was detected near southern Korean coastal waters, whereas Philippines ribotype was distributed near Ieodo and Cheju where directly affected by Kuroshio Currents. Hence, Philippines ribotype may have been originated from outer area via Kuroshio Currents. To investigate bacterial community structures followed by different C. polykrikoides bloom stages (before-, early-, middle-, late-bloom and bloom termination), cloning library analysis was used for this study since it allows the analysis of a detailed phylogenetic diversity and composition proportion of bacterial communities. In this study, α- and γ-proteobacteria were thought to be important phyletic groups during C. polykrikoides blooms in Korean coastal waters. The proportion of γ-proteobacteria which was known as the algicidal group was lowered (11.8%) at the middle of blooming phase. However, since then, Gammaproteobacteria comprised of high proportion (26.2%) when C. polykrikoides bloom was vanished. Whereas, Alphaproteobacteria showed a clear dominance during the bloom period and primary member of Rhodobacterales were abruptly increased from 37.5% (before bloom) to 73.7% (early bloom). High proportion of Rhodobacterales was maintained until late bloom stage (56%). Moreover, site Jinmok (50%) and Bangjuk-po (35.3%) where C. polykrikoides bloom occurred in October 2012 also contained high portion of Rhodobacterales. In principal component analysis (PCA), Rhodobacterales had a statistically significant relationship with C. polykrikoides bloom (P-value ≤0.001). Since, there is no ecological study about Amoebophrya sp. killing C. polykrikoides. Dinospore which is free-living form of Amoebophrya is directly related with infection rate of host cell. Although dinospore may suspected to be important to C. polykrikoides bloom termination, there is no tool to identify and quantify dinospore accurately due to its extremely small body-size (<5 μm). To investigate impacts of Amoebophrya spp. on C. polykrikoides bloom termination, qPCR assay based on copy number was developed. As results, highly significant standard curves (r2≥0.99) and one informative melting curves showed the accuracy of this quantitative assay. During this study, C. polykrikoides blooms occurred twice at site of Mulgun Harbor, in August and October of 2012. Using qPCR assay, the free-living cells showed similar dynamics to those during their parasitic stage living inside host cells. While Amoebophrya sp. 1 was predominant in August, Amoebophrya sp. 2 was strongly related with the termination of the C. polykrikoides bloom in October. Moreover, daily induced mortality reached a maximum (29.55%) a week before the C. polykrikoides bloom abruptly vanished. Accordingly, these two Amoebophrya species are thought to play an important role in terminating C. polykrikoides blooms in southern Korean coastal waters. Previous studies regarding C. polykrikoides bloom mechanisms were mainly focused on physiochemical factors, but they could not fully clarify bloom mechanisms of C. polykrikoides. Results of the present study suggested new insights of bloom mechanisms of C. polykrikoides. Ecological succession between the two ribotypes of C. polykrikoides may contribute on long bloom periods. Bacterial communities strongly interacted with C. polykrikoides blooms and Rhodobacterales may play an important role in bloom initiation. Finally, endoparasite Amoebophrya spp. was thought to be a direct trigger to mitigate C. polykrikoides bloom in Korean coastal waters.