Cyanobacterial resting cells, such as akinetes, are important seed cells for cyanobacteria’s early development and bloom. Due to their importance, various methods have been attempted to isolate resting cells present in the sediment. Ludox is a solution mainly used for cell separation in marine sediments, but finding an accurate method for use in freshwater is difficult. This study compared the two most commonly used Ludox methods (direct sediment treatment and sediment distilled water suspension treatment). Furthermore, we proposed a highly efficient method for isolating cyanobacterial resting cells and eDNA amplification from freshwater sediments. Most of the resting cells found in the sediment were akinete to the Nostocale and were similar to those of Dolichospermum, Cylindrospermum, and Aphanizomenon. Twenty times more akinetes were found in the conical tube column using the sediment that had no treatment than in the sample treated by suspending the sediment in distilled water. Akinete separated through Ludox were mainly spread over the upper and lower layers in the column rather than concentrated at a specific depth in the column layer. The mibC, Geo, and 16S rDNA genes were successfully amplified using the sediment directly in the sample. However, the amplification products of all genes were not found in the sample in which the sediment was suspended in distilled water. Therefore, 5 g to 10 g of sediment is used without pretreatment when isolating cyanobacterial resting cells from freshwater sediment. Cell isolation and gene amplification efficiency are high when four times the volume of Ludox is added. The Ludox treatment method presented in this study isolates cyanobacterial resting cells in freshwater sediment, and the same efficiency may not appear in other biotas. Therefore, to apply Ludox to the separation of other biotas, it is necessary to conduct a pre-experiment to determine the sediment pretreatment method and the water layer where the target organism exists.

Environmental DNA (eDNA) can exist in both intracellular and extracellular forms in natural ecosystems. When targeting harmful cyanobacteria, extracellular eDNA indicates the presence of traces of cyanobacteria, while intracellular eDNA indicates the potential for cyanobacteria to occur. However, identifying the “actual” potential for harmful cyanobacteria to occur is difficult using the existing sediment eDNA analysis method, which uses silica beads and cannot distinguish between these two forms of eDNA. This study analyzes the applicability of a density gradient centrifugation method (Ludox method) that can selectively analyze intracellular eDNA in sediment to overcome the limitations of conventional sediment eDNA analysis. PCR was used to amplify the extracted eDNA based on the two different methods, and the relative amount of gene amplification was compared using electrophoresis and Image J application. While the conventional bead beating method uses sediment as it is to extract eDNA, it is unknown whether the mic gene amplified from eDNA exists in the cyanobacterial cell or only outside of the cell. However, since the Ludox method concentrates the intracellular eDNA of the sediment through filtration and density gradient, only the mic gene present in the cyanobacteria cells could be amplified. Furthermore, the bead beating method can analyze up to 1 g of sediment at a time, whereas the Ludox method can analyze 5 g to 30 g at a time. This gram of sediments makes it possible to search for even a small amount of mic gene that cannot be searched by conventional bead beating method. In this study, the Ludox method secured sufficient intracellular gene concentration and clearly distinguished intracellular and extracellular eDNA, enabling more accurate and detailed potential analysis. By using the Ludox method for environmental RNA expression and next-generation sequencing (NGS) of harmful cyanobacteria in the sediment, it will be possible to analyze the potential more realistically.

목적: 이 연구는 체육 수업 상황에서 학생들이 지닌 경쟁 성향과 불링 행동의 관계를 규명하였다. 방법: 서울과 경기도 소재 중⋅고등학교 4곳에 재학 중인 학생 227명을 대상으로 중고등학생들이 인식하고 있는 경쟁 성향(협 력적 경쟁 성향과 대립적 경쟁 성향)과 불링 행동(가해 경험과 피해 경험)을 조사하였다. 수집된 자료는 기술통 계, 신뢰도, 타당도, 상관분석과 회귀분석을 실시하였다. 부가적으로 성별과 학교급에 따른 차이 검증을 진행하였 다. 결과: 분석결과, 성별에서의 경쟁 성향 차이는 확인되지 않았지만, 불링의 가해 경험은 여학생보다 남학생이 높은 것으로 밝혀졌다. 학교급에서는 중학생보다 고등학생이 대립적 경쟁 성향이 높은 것으로 나타났으며, 불링의 가해 경험과 피해 경험도 중학생보다 고등학생에게서 높은 것으로 나타났다. 협력적인 경쟁 성향은 아니지만, 대 립적인 경쟁 성향은 불링의 가해 경험과 피해 경험을 모두 예측하는 것으로 나타났다. 결론: 이러한 결과는 학생 들이 협력적 경쟁 성향이 아닌 대립적 경쟁 성향을 지닐수록 불링 행동이 유발될 수 있다는 것을 의미한다. 또 한, 중학생보다 고등학생에게서 불링 경험(가해 경험과 피해 경험)이 높다는 것을 보여준다. 경쟁 성향이 불링 행 동과 밀접하게 관련되어 있기 때문에 불링 행동을 예방 및 감소시키기 위해서 대립적 경쟁 성향이 아닌 협력적 경쟁 성향을 추구할 수 있도록 교육 및 지도해야 한다는 것을 시사한다. 이 연구가 체육 수업과 학교 현장에서 학생들의 경쟁 성향과 불링 행동을 이해하고 예방할 수 있는 중요한 기초자료가 될 것이다.