| Microplastics are a new type of pollutants in the marine environment.The accumulation of microplastics in organisms might cause various adverse reactions,thus affecting the health of marine animals.Apostichopus japonicus plays a key role in maintaining the stability of marine ecosystem.However,under the controlled environment-related concentrations,the effect of microplastic pollution on A.japonicus has not been reported.Based on the previous investigation results of microplastics in sea cucumber habitat,combined with the increasing trend of microplastic pollution,polyethylene terephthalate(PET)microplastics with particle size(0.5-45?m,2-200?m and 20-300?m)and concentration(10~3particles/kg,10~4particles/kg and 10~5particles/kg)were added to A.japonicus feed for 28 days.Then,the effects of microplastics on A.japonicus were preliminarily explored from the aspects of basic biological indicators,antioxidant effect,immune function,respiratory physiology and digestive physiology.This study provides valuable information for understanding the impact of microplastic pollution on A.japonicus,and provides a theoretical basis for studying the toxicological mechanism of microplastics on organisms.The main results of this study are as follows:(1)Effects of PET microplastics on growth and antioxidant immune system of A.japonicusAfter PET stress for 28 d,the basic biological indicators of A.japonicus were determined.The results showed that the specific growth rate(SGR),feces production rate(FPR),ingestion rate(IR)and food conversion efficiency(FCE)of A.japonicus were not affected in each concentration group(10~3particles/kg?10~4particles/kg and10~5particles/kg)of small particle size(0.5-45?m),medium particle size(2-200?m)and large particle size(20-300?m).Antioxidant enzyme activity in coelomic fluid of A.japonicus was evaluated.Superoxide dismutase(SOD)activity was not significantly affected in all treatments.The catalase(CAT)activity was significantly inhibited in the high concentration(10~5particles/kg)group with small particle size(0.5-45?m),showing a size-effect relationship and a dose-effect relationship.The smaller the particle size was,the higher the concentration was,and the more significant the effect was.The immune-related lysozyme(LZM)activity and alkaline phosphatase(AKP)activity were not significantly affected;acid phosphatase(ACP)activity showed low exposure activation at the levels of small particle size(0.5-45?m),medium particle size(2-200?m)and large particle size(20-300?m),which might be the first defense enhancement of exogenous substances in A.japonicus,but inhibited at high concentrations,indicating that the immune defense function of A.japonicus might be affected to some extent.(2)Effects of PET microplastics on respiratory physiology of A.japonicusOxygen consumption rate(OCR)was measured at 14 d and 28 d after PET stress.The results showed that small particle size(0.5-45?m),medium particle size(2-200?m)and large particle size(20-300?m)levels of each concentration group(10~3particles/kg,10~4particles/kg and 10~5particles/kg)did not affect the oxygen consumption rate of A.japonicus,indicating that the current environmental-related microplastics did not caused damage to the oxygen consumption rate of A.japonicus,and did not show a time-effect relationship.The respiratory tree tissues of A.japonicus in high concentration(10~5particles/kg)groups at each different particle sizes were further selected for non-targeted metabolomics research.By using the orthogonal partial least squares method(OPLS-DA),7,26 and 68 differential metabolites were screened out with VIP?1 and T-test P<0.05 in the OPLS-DA model as the threshold.Pathway enrichment analysis of the screened differential metabolites was carried out by KEGG database,and it was found that at large particle size(20-300?m),the metabolic disorders of arachidonic acid,alanine,aspartic acid and glutamic acid may be the potential mechanism of action of PET pollution on the respiratory physiology of A.japonicus.(3)Effects of PET microplastics on digestive physiology of A.japonicusAfter 28 d of PET stress,the activities of digestive enzymes in the intestine of A.japonicus were evaluated.The results showed that the activities of lipase(LPS)in each concentration group(10~3particles/kg,10~4particles/kg and 10~5particles/kg)at the levels of small particle size(0.5-45?m),medium particle size(2-200?m)and large particle size(20-300?m)were not significantly affected.The activities of amylase(AMS)and trypsin(TRY)under large particle size(20-300?m)were potentially negatively affected,showing a size-effect relationship and a dose-effect relationship.The larger the particle size was,the higher the concentration was,and the more obvious the effect was.In order to analyze the effect of PET on the intestinal flora of A.japonicus,the intestinal contents of A.japonicus at high concentrations(10~5particles/kg)of each particle size level were selected for high-throughput sequencing analysis.The results showed that PET microplastics did not affect the microbial richness and evenness in the intestine of A.japonicus.In addition,the composition of intestinal microflora at phylum,family and genus levels was evaluated.Only at phylum level,the abundance of Epsilonbacteraeota was significantly increased in the large particle size(20-300?m)group,and other core microflora did no significant change.It was speculated that this microplastic was not enough to disturb the intestinal homeostasis,destroy the intestinal flora and change the intestinal microbial diversity.Further untargeted metabolomics study was conducted on the intestinal tissues of A.japonicus,and 80,61 and 184differential metabolites were screened out.The results showed that under small particle size(0.5-45?m),the disorder of phosphatidylcholine metabolism might cause the damage of intestinal membrane structure and cell function of A.japonicus.Under large particle size(20-300?m),the changes of glutamine,serine,glycine,phenylalanine,tyrosine,asparagine and other metabolites might be the potential physiological mechanism of PET pollution on the intestinal antioxidant capacity and immune level of A.japonicus.In addition,its intestinal energy metabolism affected might cause energy metabolism disorders in A.japonicus. |
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