The Role Of Phosphorus In Freshwater Dinoflagellate Blooms

Dinoflagellate is a kind of eukaryotic algae widely distributed in various water ecosystem.As most species have flagella,they are capable of locomotion,they are more competitive than other immobile algae.At the same time,dinoflagellates can form cysts in unfavorable environments to avoid damage.When the environment is suitable,the cysts will germinate and return to the water.Therefore,dinoflagellate blooms are often occurred.Due to the huge threat to the natural environment,aquaculture and human health caused by dinoflagellate blooms,research on the mechanism and prevention and control strategies of dinoflagellate blooms has always been the focus of phycology and ecology.In the natural environment,the occurrence,duration and extinction stages of algal blooms are affected by ecological factors such as temperature,light,and nutrients.Phosphorus is an important component of macromolecular substances in organisms.It is involved in many key metabolic pathways and directly affects the growth of algae and the community composition of phytoplankton in the environment.Therefore,it is considered as one of the key limiting factors for phytoplankton growth in water.Dissolved phosphorus（TP）in water mainly includes soluble inorganic phosphorus（DIP）and soluble organic phosphorus（DOP）.However,the phosphorus source that phytoplankton can directly use is DIP(the main component is PO₄^3-).A large number of studies have shown that the increase of DIP leads to the occurrence of phytoplankton blooms.However,studies on dinoflagellate have found that dinoflagellate can also form large areas of water blooms in water ecosystem with low DIP concentrations.It is speculated that DOP may also play an important role in the growth and formation of dinoflagellates.However,the role of DIP and DOP in various stages of dinoflagellate blooms has not been studied,especially in freshwater dinoflagellates.Therefore,in order to explore the role of phosphorus in the process of freshwater dinoflagellate blooms,this paper intends to carry out indoor simulation of dinoflagellate bloom processes from cyst germination,growth period,population maintenance and water bloom regression,combined with in-situ water blooms in the field.Throughput sequencing,to explore the effects of phosphorus on freshwater dinoflagellate blooms from the physiological and molecular levels,aiming to reveal the role of phosphorus in freshwater dinoflagellate blooms.The main results of the study are as follows:1.The equal number of cysts were transferred to a medium containing no phosphorus（P-free）,containing inorganic phosphorus（DIP）and containing organic phosphorus（DOP:C-O-P,C-P）.The results showed that:（1）Under different phosphorus conditions,the germination of dinoflagellate cysts is different.Under P-free conditions,the germination rate of dinoflagellate cysts in the first 2-5 days was lower than that of the other three treatments with phosphorus,and the germination rate of dinoflagellate cysts under the treatment of organophosphate（C-O-P,C-P）was among the P-free group and DIP group,the germination rate of dinoflagellate cysts under DIP treatment was the highest,indicating that the presence of DIP would promote the rapid germination of dinoflagellate cysts,while phosphorus limitation would delay the germination of cysts,and organic phosphorus could compensate for phosphorus stress on cyst germination.（2）In the later stage of the experiment,the ratio of vegetative cells and the germination rate of dinoflagellate under DIP conditions were higher than that of P-free groups,while the ratio of vegetative cells and the germination rate of dinoflagellate under DOP treatment were not significant compared with P-free.The difference indicates that DIP is the best source of phosphorus for dinoflagellate cysts to maintain germination rate;（3）Under P-free conditions,more than 80%of dinoflagellate cells are in the G1 phase in the early,middle and late stages of the experiment,this indicated that the cell division of dinoflagellates was blocked in G1 phase under phosphorus stress.However,the changes of cell proportion at different stages of division could be observed under the DIP and DOP treatments,indicating that both DIP and DOP could promote the process of cell division;（4）Under different phosphorus source conditions,the different cell ratios of dinoflagellate cells in different stages may be due to the influence of different phosphorus sources on the cell division rhythm of dinoflagellate cells.C-P may change the division rhythm of dinoflagellate cells.2.The photosynthesis and growth status of dinoflagellate under different concentrations of DIP culture and different phosphorus sources（DIP,C-O-P,C-P）and the transcriptome analysis of dinoflagellate under different phosphorus sources show that:（1）With the increase concentration of phosphorus,the growth and photosynthetic efficiency of dinoflagellate are significantly enhanced,indicating that the phosphorus concentration significantly promotes the photosynthesis of dinoflagellate,and high phosphorus concentration promotes the increase of the cell density of dinoflagellate;（2）In the presence of phosphorus（DIP,C-O-P,C-P）,The specific growth rate of dinoflagellate was significantly higher than that of P-free treatment,indicating that both DIP and DOP could promote the growth of dinoflagellate,while P-free treatment significantly inhibited the growth of dinoflagellate.Under DIP treatment,the photosynthetic efficiency of dinoflagellate was significantly higher than that of P-free treatment.The fluorescence parameter results showed that DOP alleviated the effect of phosphorus stress on photosynthesis of dinoflagellate.In addition,by fitting the PC curve,the Vmax of the dinoflagellate under the DIP and DOP treatments were significantly higher than that of the P-free group,while the value of K_0.5（DIC）was significantly lower than that of the P-free group,indicating that DIP and DOP enhances the affinity of dinoflagellate for inorganic carbon;（3）Under P-free treatment,dinoflagellates are subjected to phosphorus stress,which increases the metabolism of sulfur-containing amino acids,and at the same time strengthens the transfer of ammonium,and enhances nitrogen metabolism to alleviate the stress caused by phosphorus deficiency;DIP promotes the production of NADH and the TCA process;the presence of C-O-P also enhances the decomposition of organic matter and energy metabolism of dinoflagellate;under C-P treatment,dinoflagellate stimulates the glyoxylic acid cycle,which indirectly supports the key process of energy metabolism-TCA.3.There are a variety of algae in the water.Diatoms and green algae often coexist with dinoflagellates,and diatoms and green algae can also form blooms.The maintenance of dinoflagellate populations is closely related to its competitiveness with other algae.Research and analysis on the growth trend of dinoflagellate,diatoms and green algae in monoculture and co-cultivation systems and phosphorus consumption under different phosphorus source conditions:（1）In the monoculture system,the cell density of the three kinds of algae treated by DIP and DOP was higher than that of the P-free group,indicating that all the three kinds of experimental algae could maintain their growth by using organophosphorus;（2）Among the three phosphorus-containing treatment groups,the phosphorus concentration of the DIP group decreased the fastest,indicating that DIP was still the preferred choice for algae absorption;（3）In the monoculture system,dinoflagellates absorb phosphorus most rapidly in the three kinds of algae.Even in the co-culture system,the consumption of phosphorus is faster than that of two monocultures,indicating that dinoflagellates have better phosphorus absorption capacity,They can quickly accumulate material basis for subsequent growth and reproduction,which may be an important reason for dinoflagellate to stand out in the water bloom phenomenon;（4）In the culture system,after the phosphorus concentration reduced,the growth of diatoms and green algae also decreased,while the dinoflagellate population remained at a relatively stable level,indicating that compared with diatoms and green algae,dinoflagellates are more tolerant to low phosphorus environment;（5）In the culture system,both green algae and diatoms showed a high specific growth rate when the phosphorus was sufficient in the early stage,and then the population increased rapidly.After the phosphorus decreased in the later stage,the population decreased significantly,while the growth rate of dinoflagellate was relatively weak.But it can maintain a longer growth cycle.It is speculated that dinoflagellates have different survival strategies from green algae and diatoms.Dinoflagellates are k-strategies,while diatoms and green algae are r-strategies.4.Through the observation of the dinoflagellate disappearance under different phosphorus source conditions,the results showed:（1）Under different phosphorus source treatments,the proportion of dinoflagellate dead cells in the culture system did not change significantly.The formation rate of dinoflagellate cysts in the P-free group was higher than that in the DIP and C-O-P groups in the early stage,and the cyst formation in the DIP group was still significantly lower than that in the P-free group in the later stage,indicating that under phosphorus stress,dinoflagellates formed a large number of cysts to get through the adverse growing condition,and the existence of DIP and C-O-P delay the formation of cysts;（2）In the P-free group,the contents of SOD,POD,CAT,and MDA in the dinoflagellates were significantly higher than those in the DIP and DOP groups in the later stage of the experiment,indicating that phosphorus stress aggravated the cell membrane lipid oxidation leads to an increase in the accumulation of MDA,so that the antioxidant enzyme systems such as SOD,POD and CAT are activated to cope with the intensification of intracellular oxidation.DIP,C-O-P and C-P can alleviate the peroxidation reaction to varying degrees;（3）Under phosphorus-free stress conditions,cell telomerase activity decreases,which may lead to shortening of telomeres and hinder cell replication.The presence of DIP and DOP maintains telomerase activity within a certain period of time;（4）Through the extraction of dinoflagellate DNA and the observation of DNA Ladder under different phosphorus sources,it was found that under the four phosphorus conditions,there was no DNA Ladder appeared in dinoflagellate,indicating that the dinoflagellate did not occur DNA degradation,which further verified that the dinoflagellates responded to environmental changes through the formation of cysts.5.Using transcriptome technology and combined with in-situ data to analyze the expression of genes in different periods and vertical migration of dinoflagellate blooms,the results showed that:（1）In the early bloom,the phosphorus concentration in the water is the highest,and the total phosphorus is about 0.23 mg/L;With the progress of the bloom process,the phosphorus concentration gradually decreases.In the middle of the bloom,the total phosphorus concentration is 0.1 mg/L,and it drops to 0.05 mg/L in the late bloom;（2）In the early bloom,when the phosphorus concentration is higher,the light-harvesting complex-related genes are up-regulated,and a large number of key genes of photosynthetic system II and photosynthetic system I are up-regulated,indicating that algal cells enhance photosynthesis.In the pre-bloom stage,cell cycle genes were also significantly up-regulated,indicating that algal cells have enhanced DNA replication and phosphorylation of related cell cycle kinases.The smooth progress of photosynthesis depends on the integrity of the thylakoid membrane,and sufficient phosphorus content ensures the integrity of the membrane.Moreover,phosphorus is also an important component of DNA and an essential element for the phosphorylation of cell cycle kinases.The presence of phosphorus supports a large amount of DNA replication.In addition,in the pre-bloom stage,the transcription,translation,carbon fixation,glycolysis/gluconeogenesis,and TCA pathways of algal cells were significantly up-regulated;（3）In the mid-bloom,cell movement,oxidative phosphorylation and ribosomes gene expression are enhanced;（4）In the late bloom,oxidative phosphorylation is also significantly increased,and genes related to RNA transport and translation are significantly up-regulated.The other up-regulated pathways involve ATP metabolism.Phosphorus is an essential component of ATP.Cell movement is accompanied by ATP consumption,which indirectly increases the demand for phosphorus;（4）Through the study on the diurnal vertical migration of dinoflagellates bloom,it was found that dinoflagellates concentrated in the upper water in the daytime,then dinoflagellates moved down to the deeper layer in the evening,and dinoflagellates distributed more dispersed in the evening than in the daytime.WGCNA analysis of the transcriptome of the dinoflagellate vertical migration found that the genes can be divided into 9 modules,among which the modules significantly related to phosphorus are turquoise module,black module,pink module,red module,blue module and green module,which are significantly related to phosphorus,and he core genes of these module are mainly responsible for encoding ribosomal protein and photosynthesis pathway.These core core genes have different expression patterns during the migration process.Generally speaking,dinoflagellates photosynthesize in the upper water during the daytime,and they replicate a large amount of proteins at night in the deeper water to prepare for the next day’s life activities.In summary,phosphorus plays an important role during the dinoflagellate bloom.In the early stage of water bloom,phosphorus promotes the germination and cell division of dinoflagellate cysts.During the period of rapid growth of dinoflagellate,the presence of phosphorus improves the photosynthesis and carbon assimilation ability of dinoflagellate,which obviously promotes the growth of dinoflagellate.In the population maintenance stage,the special response of dinoflagellate to phosphorus keeps the dinoflagellate population at a relatively stable level.In the process of population regression,phosphorus alleviates the peroxidation stress and delays population regression.Phosphorus participates in many important metabolic pathways,and affects the process of blooming subsidence by regulating different pathways.