Effects Of Aflatoxin B1 On Metabolism Of Procambarus Clarkii And Eriocheir Sinensis

Firstly,this study established a liquid chromatography-tandem mass spectrometry method for the determination of 8 mycotoxins in aquatic products.Methods: The samples were extracted with acetonitrile water(84:16,V:V),and purified by multifunctional immunoaffinity columns.The chromatographic column was Agilent Proshell 120 SB·C18column(2.1 mm×100 mm,2.7 μm).Mobile phase was methanol and 5 mmol/L ammonium acetate(containing 0.1% formic acid).The flow rate was 0.4 m L/min,and the column temperature was 40 ℃.The electrospray was operated in both positive and negative mode at the same time,and the samples were detected by multiple reaction monitoring mode(MRM).The linear relations of the eight mycotoxins were good within the concentration range of 1.0～50.0μLg/L(R>0.992).The limits of detection were between 0.05～0.5 μg/kg,and the limits of quantification were between 0.17～1.65 μg/kg.The average recovery rate was 75.6%～106.3%,and the relative standard deviations(RSDs)was 3.5%～9.3%.The method is simple,sensitive and accurate for the rapid analysis of mycotoxin residues in aquatic products.After injecting 400 μL of AFB1 at a concentration of 60 mg/L into the hepatopancreas of procambarus clarkii,samples were collected at 30 minutes(A30),60minutes(A60),and 90 minutes(A90)for analysis of transcriptional gene differences caused by AFB1.The results revealed a total of 399 DEGs.KEGG pathway annotation showed significant enrichment of cardiac contraction and adrenergic signaling in cardiomyocytes in all three comparisons.GO annotation revealed that the differentially expressed genes were mainly enriched in pathways such as muscle protein fibers,cellular cytoskeleton,hydrolase activity,carbohydrate catabolic process,and glucose oxidase activity.These results indicate that the adrenergic signaling pathway in the hepatopancreas of Procambarus clarkii is inhibited after AFB1 treatment,and the biological processes of carbohydrate catabolism and glucose oxidase activity play important roles.Female Eriocheir sinensis were injected with 400 μL of a 60 mg/L AFB1 solution.The hepatopancreas and ovaries were collected at 30 minutes and 60 minutes postinjection for analysis of transcriptional gene differences and metabolite differences caused by AFB1.The results revealed that,compared to the control group,210 and 250 differentially DEGs)were identified in the two experimental groups,respectively.Through NR protein annotation and GO annotation,it was found that 14 upregulated genes were associated with six functional categories: antimicrobial detoxification,ATP energy response,oxidation-reduction reaction,neural response,liver damage repair,and immune response.In the comparison between ML-C and ML-30 m,as well as ML-C and ML-60 m,a total of 228 and 401 differential metabolites DAMs were enriched in 12 pathways,including cutin,lignin,and wax biosynthesis,tyrosine metabolism,purine metabolism,nucleotide metabolism,glycine,serine,and threonine metabolism,ABC transporters,and tryptophan metabolism.Comprehensive analysis of hepatic and pancreatic metabolomics and transcriptomics revealed four significantly enriched pathways: glycine,serine,and threonine metabolism;tryptophan metabolism;tyrosine metabolism;and purine metabolism.Within these pathways,interactions between DEGs and DAMs were observed in three pathways: steroid biosynthesis,glycine,serine,and threonine metabolism,and sphingolipid metabolism.These genes and metabolites play pivotal roles in the response of crab hepatopancreas or ovaries to the invasion of AFB1.