Effects And Mechanisms Of Ocean Acidification On Paralytic Shellfish Toxins Production By Alexandrium Minutum

With the increasing global warming and marine environmental pollution,the frequent outbreak of coastal harmful algae blooms,especially marine biotoxin released by toxic algae,has caused serious impact on the health of marine ecosystem and aquaculture industry,which has attracted widespread attention worldwide.Paralytic shellfish toxins(PSTs)as a kind of marine algal toxin,are widely distributed and have obviously been affected by environmental factors.Current researches on PSTs and their toxin-producing algae mostly focus on the influence of a single environmental factor,however,multiple environmental stresses often occur in nature.The toxin-producing responses to multiple environmental stresses are lacking,and the intracellular toxin-producing mechanisms are also not clear at present.In this paper,a typical PSTs-producing dinoflagellate,Alexandrium minutum,was selected for cultivation microalgae under different CO₂ concentration levels(400 ppm,800 ppm,1200 ppm).Several important factors were detected and analyzed during the A.minutum's growth,including the production and release of intracellular and extracellular toxins,as well as related amino acids and key enzymes in the process of toxin production.The coupled effects of acidification and eutrophication on physiological responses of A.minutum were discussed in detail.The specific experimental contents were as follows:1.UPLC-MS/MS was used to establish a method for the determination of PSTs and major amino acids in A.minutum.The method is simple to operate and does not require derivatization of the sample.It can be achieved in the determination of PSTs and amino acids within 20 minutes.A.minutum contains four toxins,GTX1,GTX4,GTX2 and GTX3,and 20 major amino acids.The detection limit of 13 PSTs is between 0.1 and 8.0 ?g/L,and the limit of quantification is between 0.3 and 25.0 ?g/L;the detection limit of 20 amino acids is between 0.01 and 0.05 ?mol/L,and the limit of quantification is within the range of 0.03 to 0.20 ?mol/L.2.Under nitrogen-phosphorus ratio of 40:1,different acidification environment was set by continuous introduction of different CO₂ levels to explore the impact of ocean acidification on A.minutum growth and toxin production.Studies have shown that the increase of CO₂ concentration will promote the growth of A.minutum and the absorption of nitrogen and phosphorus nutrients to a certain extent.In the future,the marine environment under higher CO₂ concentration conditions may promote the growth of A.minutum.The increase of CO₂ concentration can promote the production of PSTs in A.minutum.The content of four kinds of PSTs in the intracellular and extracellular culture medium also increased,and the content of PSTs in the extracellular medium increased to a higher degree.At the same time,the increase of CO₂ concentration will promote the oxidation of A.minutum's less toxic GTX2 and GTX3 to the more toxic GTX1 and GTX4,resulting in an increase in A.minutum's toxicity,while in the extracellular medium,the increase of CO₂ concentration also changed the dominant position of GTX2 and GTX3,but the change was less than that in the intracellular medium.3.The arginine,acetic acid,and adenosylmethionine synthesize PSTs under the action of enzymes.In this study,several important amino acids including arginine,ornithine,proline,etc.,as well as key enzymes related to the toxin synthesis process such as argininosuccinate synthase,nitrate reductase,aspartate aminotransferase,etc.were selected for analysis and determination,to explore the regulatory mechanism of A.minutum's response to toxin production under the stresses of eutrophication and acidification.The results show that the content of various amino acids in A.minutum cells is quite different,and the fluctuation of PSTs is obviously lagging behind that of each amino acid.During the growth cycle of A.minutum,the content of enzymes per unit volume of the culture medium does not change much,but it can still catalyze the decomposition,transformation and synthesis of a large number of amino acids and other substances.The content and consumption of different enzymes under the condition of CO₂ concentration of 800 ppm are significantly higher than the other two groups.In this paper,an analytical method for paralytic shellfish toxins and main amino acids in toxic dinoflagellate is established.It is easy to operate and has a low detection limit.It can fully meet the analysis on relevant toxins and amino acids in the indoor culture experiments of A.minutum.Through indoor culture experiments under controlled conditions to simulate the acidification environment of seawater under eutrophication conditions,and explore the response law of A.minutum under different CO₂ concentration conditions,the experimental results show that the increase of CO₂ concentration can promote growth and toxin-producing of A.minutum to a certain extent.A.mintum will be more toxic in the ocean acidification environment under eutrophication conditions.The regulation mechanism of A.minutum's intracellular toxin production is explored.The intracellular amino acids in toxic A.minutum are continuously synthesized,decomposed,and transformed through the citric acid cycle and the urea cycle.Finally,arginine,acetic acid,and adenosylmethionine are in the enzyme Synthesize PSTs under the action.