Microplastic and Copper Exposure Effects on Intestinal Gene Expression in Eriocheir sinensis: A Transcriptomics-Based Investigation

Over 80% of marine litter is composed of plastic waste, which reaches the oceans via atmospheric transport, surface runoff, and human activities such as shipping and fishing. These plastics degrade into microplastics (MPs)—prevalent environmental pollutants < 5 mm, which possess a "carrier effect", enabling them to adsorb contaminants such as heavy metals. CuSO4—frequently used in aquaculture to manage diseases and cyanobacterial blooms—can introduce excess Cu2+ into aquatic environments, adversely affecting water quality and aquatic life. The Chinese mitten crab (Eriocheir sinensis) is vital to freshwater aquaculture in China. Mature E. sinensis migrate to the Yangtze River estuary annually for reproduction; however, estuarine ecosystems are becoming 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 MP or Cu2+ effects on E. sinensis independently, however, their combined effects remain underexplored. Here, MPs (0.4 mg/L) and Cu2+ (0.1 mg/L) were selected as the experimental concentrations with four treatment groups: Group M (0.4 mg/L MP-exposed); Group C (0.1 mg/L Cu2+-exposed); Group MC (0.4 mg/L MP + 0.1 mg/L Cu2+ combined-exposed; and Group D (blank control group) to investigate the effects of MPs and Cu2+ on the intestinal tract of E. sinensis after 21 days of single and combined exposure. Transcriptome sequencing of the 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. In total, 109, 644 transcripts were identified, with 68, 005 exceeding 1800 bp in length. Differential expression analysis revealed 1, 650 and 1, 874, 3, 797 and 1, 073, and 1, 492 and 1, 305 upregulated and downregulated differentially expressed genes (DEGs) in Groups M, C, MC, respectively. Notably, DEGs associated with antioxidant defense, immune response, and energy metabolism differed significantly among the comparison groups. Catalase (cat) and peroxiredoxin (prdx) were downregulated, whereas trim, toll-like receptor (tlr) and complement component 1 (c1) were upregulated in the immune system. Cytochrome P450 (cyp450) was significantly downregulated across the treatment groups. Energy metabolism DEGs also varied, with carbonic anhydrase (ca) up-regulated and vesicular adenosine triphosphatase (v-atpase) down-regulated. KEGG pathway enrichment analysis indicated that in Groups M vs D, 1, 594 DEGs in the intestine were mapped to 333 pathways. Among these, 34 pathways were significantly enriched, including oxidative phosphorylation and glutathione metabolism. In Groups C vs D, 2, 445 DEGs in the intestine were mapped to 340 pathways. Among these, nine pathways were significantly enriched, primarily involving DNA replication and ABC transporters. In Groups MC vs D, 1, 198 DEGs in the intestine were mapped to 326 pathways. Among these, 14 pathways were significantly enriched, including the complement and coagulation cascades and metabolism of xenobiotics by cytochrome P450. The DEGs were predominantly enriched in pathways related to oxidative phosphorylation, glutathione metabolism, xenobiotic metabolism by cytochrome P450, and ABC transporters. These findings indicate that both individual and combined exposure to MPs and Cu2+ disrupts the antioxidant, immune, and energy metabolic systems of E. sinensis. The glutathione metabolic pathway was particularly inhibited in Groups M, C, and MC. MPs and Cu2+ may affect the expression of cyp450 and related genes (ugt), potentially compromising the immune function of E. sinensis. Notably, ugt was significantly upregulated in Groups M vs D and downregulated in Groups MC vs D. The number of DEGs linked to the oxidative phosphorylation pathway varied across comparisons, with 41 DEGs in Groups M vs D and seven in Groups C vs D and MC vs D being enriched in this pathway. MPs may affect the oxidative phosphorylation pathway by inhibiting the expression of cox and atpase genes. In contrast, exposure to Cu2+ alone and co-exposure to MPs had comparatively smaller impacts on this pathway. The mechanisms underlying the stress response of E. sinensis to MPs and Cu2+ exposure were further elucidated in this study. The results of this study revealed the mechanisms of MPs and Cu2+'s effects on the antioxidant, immune defence, and energy metabolism of Eriocheir sinensis, which provides an important theoretical basis for the study of E. sinensis culture and environmental toxicity.