Microplastics (nanoplastics) pollution has been a major ecological issue threatening global aquatic ecosystems. However, knowledge of the adverse effects of nanoplastics and the effects on freshwater ecosystems is still limited. To understand the impacts of nanoplastics on freshwater ecosystems, it is essential to reveal the physiological changes caused by nanoplastics in freshwater organisms, especially at their early life-history stages. In the present study, the larval channel catfish Ietalurus punetaus were exposed to gradient concentrations (0, 5, 10, 25 and 50 mg/L) of 75-nm polystyrene nanoplastics (PS-NPs) for 24 h or 48 h, and changes in contents of energy metabolites, metabolic enzyme activities and transcriptome were assessed. The results showed glucose and triglyceride contents increased after 24 h of exposure to 10 or 25 mg/L of PS-NPs but decreased with increased concentrations or prolonged exposure duration. Activities of most metabolic enzymes analyzed decreased in the larvae after 48 h of exposure, especially in 25 or 50 mg/L of PS-NPs. These suggested that PS-NPs caused huge energy consumption and disturbed the energy metabolism in larval fish. Transcriptomic analysis showed that 48 h of exposure to 50 mg/L PS-NPs affected the expression of genes involved in protein digestion and induced response of proteasomes or heat shock proteins in the larval I. punetaus. The genes involved in peroxisome proliferator-activated receptors (PPAR) pathway and biosynthesis of amino acids were activated after the exposure. PS-NPs also depressed the expression of the genes involved in gonad development or muscle contraction in the larval I. punetaus. Overall, acute exposure to 75-nm PS-NPs disrupted the energy metabolism by consuming  the energy reserves, and affected a series of molecular pathways which may further affect the development and survival of fish. This study provided the information about adverse effects of nanoplastics on the fish larvae and revealed the molecular pathways for the potential adverse outcomes.

The channel catfish Ietalurus punetaus larvae were obtained from the National Genetics and Breeding Center of Channel Catfish (Nanjing, P.R. China) and were reared in tanks with aerated tap water at 26^oC, a dissolved oxygen concentration of about 5.0 mg/L and a 12 h light/dark photoperiod. After two-week acclimation and a 24-h fasting, the larvae were exposed to 50 mg/L of 75 nm polystyrene nanoplastics for 48 h plus a control group without exposure. The unlabeled 75-nm PS-NPs particles used in the present study were purchased from Baseline Chromtech Research Centre, Tianjin, China. After the exposure, the larvae were whole-body sampled and the RNAseq was performed. Each group contained three replications.

Total RNA was isolated from the whole-body samples using a miRNA isolation kit (Invitrogen, USA). The concentration and integrity of total RNA were determined with a bioanalyzer (Nanodrop 2000, Thermo Scientific, USA) and agarose gel electrophoresis, respectively. The mRNA was isolated from total RNA using oligo(dT) magnetic beads and then broken into fragments. Double-stranded cDNAs were generated with reverse transcribed fragments followed by end repair and adapter attachment. After PCR amplification, the quality of the cDNA library was evaluated using an Agilent bioanalyzer (Agilent, USA) and then paired-end sequenced on the Illumina Hiseq X Ten platform.

Clean reads were obtained by removing reads containing poly-N and with low quality using Trimmomatic (v. 0.36). Clean reads were mapped to a reference genome using hisat2 (v. 2.2.1.0). Expression levels of genes were assessed as fragments per kilobase of exon million (FPKM) using Cufflinks (v. 2.2.1). For genes from the PS-NPs group and control group, FPKMs of each gene were used to conduct DEGs analysis with DESeq R package (v. 1.18.0). The thresholds for scanning DEGs were set as P-adjust < 0.05 and fold change > 2.