Research On The Metabolism Of PSTs In Crassostrea Gigas Using By ¹⁵N Stable Isotope

Paralytic shellfish toxins?PSTs?produced by toxic algae in Harmful algal blooms ?HABs?are widely distributed and can be accumulated in various nutritional marine organisms through the food chain.After being consumed by humans,PSTs can interact with sodium channel to hinder nerve conduction and cause paralytic poisoning,which can cause sub-lethal or lethal effects on humans.Marine bivalve molluscs are the main carriers by filtering large amounts of toxic algae and enriching toxins.To clarify the relationship between PSTs and bivalve further,we chose Alexandrium minutum as PSTs producer and oysters Crassostrea gigas as PSTs carrier.In this paper,the effect of nanoparticles?NPs?exposure on A.minutum was observed and ¹⁵N stable isotope was used to label PSTs in A.minutum.Then,Crassostrea gigas were fed with ¹⁵N-A.minutum for PSTs accumulation.The distribution and transformation of PSTs in the shellfish were studied and the viscerals were collected for non-targeted metabolomics to determine the differential metabolites and stress-related pathways related to PSTs metabolism in bivalve.The main findings are as follows:?1?The four NPs?nTiO₂,nZnO,nSiO₂,and nC₃N₄?showed different effects on the growth,physiological changes,and PSTs production of A.minutum.nTiO₂ showed the lowest growth inhibition and nZnO showed the most obvious inhibition.Reacting to NPs,reactive oxygen species?ROS?and malondiadehyde?MDA?were produced,consequently with the similar changes of superoxide dismutase?SOD?and catalase?CAT?.The largest ROS production was induced by nC₃N₄,followed by nZnO,nSiO₂,and nTiO₂.Compared with the control group,the PSTs toxicity per cell decreased by 28.93%,68.99%,53.17%and 62.67%when exposed to 5 mg/L nTiO₂,nZnO,nSiO₂ and nC₃N₄ and increased by 6.25%when exposed to 50 mg/L nTiO₂.However,the proportion of GTX1/4,the more toxic derivatives,increased when unicellular toxicity decreased.The change of PSTs profile might be biological adaptation for A.minutum to overcome the stress of NPs exposure.?2?The A.minutum was cultured in f/2 medium with ¹⁵N-NaNO₃ instead of ¹⁴N-NaNO₃ as nitrogen source to obtain the ¹⁵N-A.minutum.Both algae cells and crude toxin extracts were analyzed for isotopic abundance changing,it was found that the ¹⁵N abundance in the cells was much higher than that in PSTs extracts until 28^th generation(reaching the ideal ¹⁵N abundance:?90%).PSTs contains 6-7 nitrogen atoms in its molecular structure so that it should take more time for PSTs being fully labeled with ¹⁵N than other components in the cell.Besides,the effects of trace nitrogen in seawater and reagents used in experiment may be another reason for the difference.After feeding on ¹⁵N-A.minutum,the oysters showed toxin accumulation.The toxins that had been labeled in the algae were detected in the oyster tissue and were transformed from GTX1-4 to dcGTX1-4and M4.?3?According to previous researches,visceral mass is the main tissue for PSTs accumulation and transformation in oyster.Therefore,visceral masses were the research subjects for metabolomics analysis in our study.After UPLC-MS analysis,single and multiple variable analysis,the TOP10 differential metabolites screened were pantothenic acid?PA?,levan,1-nitronaphthalene-5,6-oxide,N-acetyl-D-glucosamine 6-phosphate,phthalic acid,oxidized glutathione?GSSG?,?-alanyl-L-arginine,PC?20:5?5Z,8Z,11Z,14Z,17Z?/P-18:0?,PE?20:3?8Z,11Z,14Z?/18:0?,SM?d18:0/20:2?11Z,14Z??.The related metabolic pathways analyzed through Kyoto Encyclopedia of Genes and Genomes?KEGG?ID of differential metabolites were?-Alanine metabolism,Glycosylphosphatidylinositol?GPI?-anchor biosynthesis,Autophagy,Glycerophospholipid metabolism,Sphingolipid metabolism,Linoleic acid metabolism,Pantothenate and CoA biosynthesis,Starch and sucrose metabolism,Glutathione metabolism and so on.These different metabolites and pathways are closely related to inflammatory response,oxidative stress,lipid metabolism and carbohydrate metabolism in oysters.