| Microplastics (MPs) are particles smaller than 5 mm and are prevalent environmental pollutants. Over 80% of marine litter is composed of plastic waste, which reaches oceans via atmospheric transport, surface runoff, and human activities such as shipping and fishing. These plastics degrade into MPs, which possess a "carrier effect", enabling them to adsorb contaminants like heavy metals. Copper sulfate (CuSO4), frequently used in aquaculture to manage diseases and cyanobacterial blooms, can introduce excessive copper ions (Cu2+) into aquatic environments, adversely affecting water quality and aquatic life. The Chinese mitten crab (Eriocheir sinensis) is a vital species in China's freshwater aquaculture. Annually, mature E. sinensis migrate to the Yangtze River estuary for reproduction. However, the estuarine ecosystem is increasingly disturbed, making mitten crabs vulnerable to MPs and heavy metal contamination. The intestinal tract, which directly interacts with ingested pollutants, is particularly susceptible. Previous studies have examined the effects of MPs or Cu2+ on E. sinensis independently, but the combined impact remains underexplored. In this study, 0.4 mg/L MPs and 0.1 mg/L Cu2+ were selected as the experimental concentrations, and four treatment groups were set up: 0.4 mg/L MPs-exposed group (Group M), 0.1 mg/L Cu2+-exposed group (Group C), 0.4 mg/L MPs + 0.1 mg/L Cu2+ combined-exposed group (Group MC), and blank control group (Group D) to investigate the effects of MPs and Cu2+ on the intestinal tract of E. sinensis after 21 days of single and combined exposure. After 21 days, transcriptome sequencing of intestinal tissues was conducted using Illumina's high-throughput platform, generating 197,908,972 raw reads. Post-quality filtering yielded clean reads across the four groups, with 94.71%-95.48% Q30 scores. 109,644 transcripts were identified, with 68,005 exceeding 1800 bp in length. Differential expression analysis revealed 1,650 up-regulated and 1,874 down-regulated DEGs in Group M, 3,797 up-regulated and 1,073 down-regulated DEGs in Group C, and 1,492 up-regulated and 1,305 down-regulated DEGs in Group MC. Notably, DEGs associated with antioxidant defense, immune response, and energy metabolism exhibited significant differences among comparison groups. In particular, catalase (cat) and peroxiredoxin (prdx) were down-regulated, while trim, Toll-like receptor (tlr), and complement component 1 (c1) were up-regulated in the immune system. Cytochrome P450 (cyp450) showed significant down-regulation across treatment groups. Energy metabolism DEGs also varied, with carbonic anhydrase (ca) up-regulated and vesicular adenosine triphosphatase (v-atpase) down-regulated. KEGG pathway enrichment indicated that, in M vs. D, 1,594 DEGs in the intestine were mapped to 333 pathways. Among these, 34 pathways were significantly enriched (p < 0.05), including oxidative phosphorylation and glutathione metabolism. In C vs. D, 2,445 DEGs in the intestine were mapped to 340 pathways. Among these, 9 pathways significantly enriched (p < 0.05), primarily involving DNA replication and ABC transporters. In MC vs. D groups, 1,198 DEGs in the intestine were mapped to 326 pathways. Among these, 14 pathways were significantly enriched (p < 0.05), including the complement and coagulation cascades, metabolism of xenobiotics by cytochrome P450, among others. The DEGs were predominantly enriched in pathways related to oxidative phosphorylation, glutathione metabolism, metabolism of xenobiotics by cytochrome P450, and ABC transporters. The findings indicate that both individual and combined exposures to MPs and Cu2+ disrupted the antioxidant, immune, and energy metabolism systems in E. sinensis. The glutathione metabolism pathway was particularly inhibited in the M, C, and MC groups. MPs and Cu2+ may impact the expression of cytochrome P450 and related genes (cyp450, ugt), potentially compromising the immune function of E. sinensis. Notably, ugt was significantly upregulated in M vs. D and downregulated in MC vs. D. The number of DEGs linked to the oxidative phosphorylation pathway varied across comparisons, with 41 DEGs in M vs. D, 7 in C vs. D, and 7 in MC vs. D being enriched in this pathway. MPs might affect the oxidative phosphorylation pathway by inhibiting the expression of cox and atpase genes. In contrast, exposure to Cu2+ alone, as well as co-exposure with MPs, had a comparatively smaller impact on this pathway. The mechanisms underlying the stress responses of E. sinensis to MPs and Cu2+ exposure are further elucidated in this study. |
