Physiological And Biochemical Mechanism Of Modified Clay Controlling Harmful Algal Blooms

In recent years,the occurrence and scale of harmful algal blooms（HABs）have increased gradually.HABs have become a worldwide marine disaster.At present,modified clay（MC）application is considered as one of the most effective strategies from varieties of methods for controlling HABs.In fact,MC application has been widely applied to large-scale treatment of HABs and has become the standard method for the emergency treatment of HABs in China.Many field applications have revealed that a removal efficiency（RE）of 80%is sufficient to control the HAB,although considerable numbers of residual algae are thought to be able to re-grow into a HAB within a short time,re-proliferation of the residual algae has rarely been observed.In the present study,to gain insight into the mechanism underlying the growth inhibition of residual microalgae,the typical harmful dinoflagellates——Amphidinium carterae Hulburt and Karenia mikimotoi were treated with different concentrations of MC,and then the oxidative stress system and photosynthesis of the residual A.carterae and K.mikimotoi were measured.Furthermore,real-time fluorescent quantitative PCR（RT-qPCR）was used to study the transcript levels of genes related to stress adaptation and photosynthesis in the residual K.mikimotoi.The main results were as follows:（1）MC effectively removed A.carterae,and the growth of the residual algae was obviously inhibited when the MC dosage was equal to or higher than 0.10 g/L.Further investigation suggested that excessive reactive oxygen species（ROS）accumulated in the residual algae,indicated by increased malondialdehyde（MDA）contents.There were significantly negative correlations between the residual algal density and superoxide dismutase（SOD）activity,catalase（CAT）activity and MDA content,which directly indicated the accumulation of intracellular ROS.The excessive levels of ROS in residual algae might be the main internal factor inhibiting their growth.In addition,the pigment contents and net photosynthetic rate were decreased after treatment,indicating that the photosynthetic efficiency was severely decreased.Moreover,compared with the control,the active reaction centers（RCs）per excited cross section（RC/CS₀）decreased,indicating that the partial RCs became inactivated.Therefore,the residual activated RCs were over-excited,suggested by the increases in the absorption flux per photosystem II（PSII）RC（ABS/RC）,the trapping flux per RC（TR₀/RC）and the electron transport flux per RC（ET₀/RC）;while the TR₀/ABS and ET₀/ABS decreased,indicating MC led to an imbalance between photosynthetic light absorption and energy utilization.After adding 0.25 g/L MC at 3 h,the W_K and V_J of the residual microalgae increased to 1.24 and 1.20 times that of the control,respectively,indicating that the oxygen-evolving complex activity was impaired,the electron transport chain（ETC）from the primary quinone electron acceptor Q_A to the secondary quinone electron acceptor Q_B was blocked.These changes lead to large accumulation of ROS at PSII.（2）K.mikimotoi,as the second dominant harmful algal bloom specise in China,was treated with different concentrations of MC and its changes of growth were studied.After treatments with 0.50 g/L MC,the RE of K.mikimotoi was 64%,and the growth of residual microalgae was significantly inhibited.Significant increases in the hydrogen peroxide（H₂O₂）content and MDA content were observed after MC treatment,indicating that MC induced significant oxidative stress in the residual K.mikimotoi.In addition,the increased ROS level was hypothesized to be the main internal factor inducing the inhibition of residual algae growth,as indicated by the significantly negative correlations between the residual algal density and H₂O₂ content.Further investigation showed that although the achieved overall photosynthetic efficiency was not significantly affected by MC,and that the light reaction of photosynthesis could provide sufficient nicotinamide adenine dinucleotide phosphate（NADPH）and transmembrane proton gradient required for the synthesis of adenosine triphosphate（ATP）,MC damaged the photosynthetic apparatus,as indicated by reduction in the maximal photochemical efficiency of PSII（F_v/F_m）and the performance index(PI_ABS).The absorption energy per unit RC increased,but the capacity for energy utilization decreased,ETC was blocked,and the Q_A^-was accumulated,leading to the generation of ROS in chloroplast.（3）The stable endogenous reference wasβ-actin for RT-qPCR analysis of K.mikimotoi after treatmets with MC.And to gain insight into the effects of MC on the physiological activities from the level of genes,the transcript levels of genes related to stress adaptation and photosynthesis in residual K.mikimotoi after treatment with 0.50g/L MC were determined.The results showed that heat shock protein 90（HSP90）and HSP70 were rapidly induced in the stress response of residual algae after MC treatment and were important regulators of the stress resistance process.The changes in their encoding genes（hsp90 and dnaK）expression were significantly correlated with the expression of the most photosynthetic genes,indicating their potential involvement in photosynthesis repair in K.mikimotoi.MC significantly inhibited the expression of six key protein genes（psbC,psbB,psbA,psbD,psaB and psaA）in the light reaction in residual K.mikimotoi,indicating that photosystem II（PSII）and PSI were damaged to a certain extent.However,this damage did not affect the actual photochemical efficiency of the light reaction.Over time,under the tight regulation of signal transduction,the expression of the 6 proteins increased to different degrees on the second day.The transcript level of genes encoding ATP synthase in residual K.mikimotoi was significantly decreased at 3 h and 1 d,and this decreased level continued to be observed until the end of the experiment,which suggested that utilization of the transmembrane proton gradient provided by the light reaction may decrease and that the process of ATP synthesis may be reduced.Although the transcript level of the ribulose-1.5-bisphosphate carboxylase/oxygenase large subunit gene（rbcL）was decreased significantly at 3 h,it recovered rapidly over time,and it is reasonable to consider that the Rubisco protein content was not the limiting factor in photosynthesis.However,changes in CbbX（a red-type Rubisco activase）and ATP synthase expression suggested that the activity of Rubisco may be reduced,which would directly affect the efficiency of CO₂ fixation,inevitably induce a large accumulation of ROS,and affect the normal growth.In summary,the effect of MC on the growth of residual microalgae was studied from the perspective of physiological and biochemical characteristics.The results showed that MC induced the accumulation of excessive ROS,and the oxidative stress was the main inhibitor of growth.MC also exerted great influence on photosynthesis,destroyed the photosynthetic apparatus,disordered the balance in light energy absorption and utilization,and hindered ETC,which may lead to the generation of ROS in chloroplast.Furthermore,molecular biology method was also used to revel the photosynthetic activities of residual algae.