升温与聚苯乙烯微塑料复合暴露对长牡蛎血细胞功能、免疫基因表达和能量代谢的影响

Combined Effects of Elevated Temperature and Polystyrene Microplastics on Hemocyte Function,Immune-Related Gene Expression,and Energy Metabolism of Crassostrea gigas

为阐明全球气候变暖和微塑料复合胁迫对长牡蛎(Crassostrea gigas)免疫应答、氧化应激和能量代谢的影响,本研究采用3个微塑料(microplastics,MPs)水平[无微塑料、小粒径聚苯乙烯微塑料(SPS-MPs,6μm)和大粒径聚苯乙烯微塑料(LPS-MPs,50~60μm)]和2个温度水平(20℃和25℃)对长牡蛎进行了为期21 d的单一和复合暴露,检测分析了各组长牡蛎血细胞功能[吞噬活性、活性氧(reactive oxygen species,ROS)含量]、糖原含量以及免疫相关基因表达的变化。研究结果表明,SPS-MPs暴露能增加长牡蛎血淋巴细胞中ROS含量,降低血细胞吞噬活性,揭示SPS-MPs毒性作用更强。升温与微塑料的协同作用增加了长牡蛎消化腺组织中的糖原含量。实时荧光定量PCR结果显示,升温与SPS-MPs复合暴露组长牡蛎消化腺组织通过上调热休克蛋白90(heat shock protein90,HSP90)、核因子κB抑制蛋白(inhibitor of NF-κB,IκB)和p53基因表达量进行免疫应答;升温与微塑料的拮抗作用增加了鳃组织p53基因表达量,揭示p53基因参与了鳃组织免疫调控。总之,升温与微塑料复合暴露能影响长牡蛎的氧化应激、免疫反应和能量代谢,升温与SPS-MPs长期暴露可能对长牡蛎的种群维持造成负面影响。

Bivalves are affected by various stressors,such as global warming and microplastics,in the marine environment.Microplastics are one of the most concerning pollutants worldwide,and high seawater temperatures caused by global warming influence the survival of marine organisms.However,little is known about the combined effects of elevated temperature and microplastics(MPs)on marine organisms,and most studies conducted in recent years have investigated the two factors,respectively.Thus,it is necessary to investigate the combined effects of elevated temperature and MP exposure on marine life.The Pacific oyster Crassostrea gigas is a widely distributed marine mollusk,and has very important economic value.The aim of the current study was to explore the toxic effects of elevated temperature and microplastic co-exposure on the hemocyte function,immune-related gene expression,and energy metabolism of C.gigas.In the current study,oysters were exposed to three levels of microplastics(no microplastics,6μm microplastics:SPS-MPs,and 50~60μm microplastics:LPS-MPs)and two temperature levels(20℃and 25℃)for 21 days,and the phagocytosis rate and reactive oxygen species(ROS)content of hemocytes,glycogen content in digestive glands,and immune-related gene expression in digestive glands and gills were examined on the 21st day.2',7'-Dichlorodihydrofluorescein diacetate and fluorescent microspheres were used to measure the ROS content and phagocytosis ratein hemocytes of C.gigas by flow cytometry,respectively.The glycogen content was measured using detection kits.Total RNA was isolated by TRIzol reagent,and the concentration was measured by Nanodrop.M-MLV Reverse Transcriptase was used for cDNA synthesis.The expressions of immune-related genes[inhibitor of NF-κB(IκB),p53,and heat shock protein 90(HSP90)]were examined by quantitative real-time PCR in the digestive glands and gills of oysters from each treatment group.Two-way ANOVA was used to analyze the interactive effects of elevated temperature and microplastics on tested parameters of oysters using SPSS software.The results showed that exposure to SPS-MPs could elevate ROS content and reduce phagocytosis in hemocytes,but no significant interaction was found between elevated temperature and microplastic effects on ROS content and phagocytosis rate in hemocytes(P>0.05).The 25℃+LPS-MPs exposure significantly decreased phagocytosis in hemocytes compared with single LPS-MPs and elevated temperature exposures(P<0.05).Single SPS-MPs exposure significantly decreased phagocytosis in hemocytes compared with single LPS-MPs exposure(P<0.05).In digestive glands,there was a significant interaction between elevated temperature and microplastics in glycogen content(P<0.05),and the combined exposure could increase the glycogen content compared with other treatments.In digestive glands,the 25℃+LPS-MPs exposure significantly increased glycogen content compared with single elevated temperature and single LPS-MPs exposure(P<0.05).In digestive glands and gills,there was a significant interaction between elevated temperature and microplastics in the expressions of HSP90,IκB,and p53 genes(P<0.05).The 25℃+SPS-MPs exposure significantly upregulated the expression of HSP90,IκB,and p53 genes in the digestive glands of oysters compared with single SPS-MPs and single elevated temperature exposures(P<0.05).The 25℃+SPS-MPs exposure significantly downregulated the expression of the HSP90 gene in the gills of oysters compared with single SPS-MPs exposure(P<0.05).Single elevated temperature and single microplastics exposure significantly upregulated the expression of the IκB gene compared with the control in gills(P<0.05).The combined exposure of elevated temperature and microplastics showed a significant antagonistic effect on the expression of the p53 gene in gills.Microplastics exposure downregulated p53 gene expression compared with the control at 20℃,while it upregulated p53 gene expression compared with single elevated temperature at 25℃.These results indicated that the p53 gene plays an important role in regulating the immune response in both digestive glands and gills.The interaction between elevated temperature and microplastics on the mRNA expression of HSP90 and IκB genes in digestive glands of C.gigas was size-dependent:A synergistic effect was found between SPS-MPs and elevated temperature,and an antagonistic effect was found between LPS-MPs and elevated temperature.A significant antagonistic effect was found between elevated temperature and microplastics on the mRNA expression of the IκB gene in gills,and the regulation pattern was different from that in the digestive glands,indicating that the regulation effect of the IκB gene was tissue-specific.In conclusion,the combined exposure of elevated temperature and microplastics can increase the glycogen content in the digestive glands of C.gigas,induce an immune response in digestive glands and gills,and trigger the oxidative stress response in hemocytes.Microplastics can cause stronger oxidative stress in hemocytes than elevated temperature.Moreover,a significant interactive effect was found between elevated temperature and microplastics on glycogen content in digestive glands and the expression of immune-related genes(HSP90,p53,and IκB)in digestive glands and gills.The results of this study provide valuable information for evaluating the toxic effects of microplastics on marine organisms under a global warming background.