Will High Osmolality In Seawater Cause DNA Breaks In Chlamys Farreri?

Not a lot of studies have already been made in the osmotic effects on terrestrial life’DNA, let alone marine life, so this research is in the frontier of related studies. In thisresearch, Comet Assay and TUNEL assay are used in the primary Chlamys farrerihemolymph culture, and a phenomenon has been shown that high osmotic pressure ofthe seawater (1200mOsm/L) will cause Chlamys farreri’s DNA fragmentation,producing3’-OH ends. And this DNA fragmentation is reversible when the osmolarityof external environment is adjusted to the low osmolarity levels (460mOsm/L), whenDNA breaks will be automatically repaired quickly.1. Primary Chlamys farreri Blood Cells Culture.Chlamys farreri blood cells is used for primary culture. The improved L-15mediumof1200mOsm/L is uesd as basic medium, and cuture are under20℃, pH=7.6.Cultured blood cells within1~3days will adhere to petri dish widely, and the bloodcells’ viability rate is higher than90%. After10~15days’ culturing, the cells graduallydie. The first3days’ cells in culture fully meet the Comet Assay&TUNEL assay’sstandards.2. Comet AssayComet Assay is carried on the base of primary Chlamys farreri blood cell culture, anduse logarithmic growth phase H1299cells as negative control. It is found after32hcontinuous monitoring that, blood cells in low osmolality (460mOsm/L) always havea much lower Tail DNA%than the ones in high osmolarity (1200mOsm/L), theformer is in the range of6.04%~13.29%, and the latter gradually increase from28.75%to60.86%. After the exchange of osmotic pressure, the Tail DNA%situation in high and low osmolarity also exchange. Therefore, a conclusion could be reachedthat high osmotic pressure will cause DNA breaks in Chlamys farreri blood cells,when osmotic pressure is reduced, DNA breaks will be repaired and reduced. Besides,in vivo blood cells Tail DNA%from different Chlamys farreri individuals are alsoexamined. It is found that their Tail DNA%changes over time. Sometimes it is low, atabout10%or less, Sometimes it is relatively high, at about25%or more;3. TUNEL AssayTUNEL Assay is carried on primary Chlamys farreri blood cells culture,too. It isfound that under the excitation wavelength of465-495nm, the blood cells in highosmotic pressure have evident and strong green fluorescence, this show that they havesignificant DNA fragmentation. While the cells in low osmotic pressure have weakand unconspicuous green fluorescence, this show that it has little DNA fragmentation.After exchange of osmotic pressure, the green fluorescence situation exchangescorrespondingly. Hence, the conclusion could be solidified again by TUNEL Assaythat high osmotic pressure will cause DNA breaks in the Chlamys farreri blood cells,when osmotic pressure is reduced, DNA breaks will be repaired and reduced.What’s the biological significance of the DNA breaks in Chlamys farreri caused byhigh osmotic pressure? Mollusk has already adapted to the high osmotic seawatersince they were born, so will or will not these DNA breaks be repaired in their dailylife? These question are still to be resolved.