## 2 Materials and Methods
### Experimental Design and Sampling  
The materials of virgin MPs were fibers and disposable plastic cups, which were purchased from a local store (Guangzhou, China). Materials were identified through a Fourier transform infrared spectrometer (FTIR Spectrometer, Vertex 70, Bruker) and found to be polypropylene (PP) (Supplementary Figure S1). Plastic cups were cut into pieces by surgical scissors and then filtered by a 5 mm steel screen. These fragments made from plastic cups were 3-4 mm. The virgin fibers themselves were also 3-4 mm in length. The majority of microplastics (more than ninety percent) used in this study were 3-4 mm through measured statistically.  
After, we ensured the materials, processed fragments and virgin fibers were added into fish feed; Supplementary Figure S2 shows the process of making the feed contained MPs. Specifically, the commercial feed was soaked in water to make a dough, then 0.3% weight MPs were added into the dough according to a previous protocol [21]. The feed pellets were 2-5 mm in size and 3.33 g/kg of MPs in concentration, which mean there were 3.33 g fibers and fragments in 1 kg feed. Therein, 3.33 g MPs contained 1.665 g fibers and 1.665 g fragments. After being dried in air for 48 h, the pellets were stored in airtight bags until use. The control group of feed was treated in the same way above, but without the addition of MPs.  
The concentration of MPs set in the water was 1 mg/L at environmentally relevant level [2,3]. Fibers and fragments represented 50%. Specifically, 90 L water contained 45 mg fibers and 45 mg fragments. MPs were thoroughly mixed in the water before fish put in. The water was refreshed every week, and MPs in water were supplemented at the same time. Fifty percent of water was changed.  
Four groups were divided according to whether the MPs were added in water or in feed. In particular, MPs were found in both feed and water in Group A. MPs were found only in water and not in feed in Group B, while in Group D, MPs were found only in feed not in water. Group C is the control group, and there was no MPs in feed or water.  
The fish were cultured for 28 d in such setting groups, starting from 28 June 2020. The culture conditions were: 12 h light, 12 h dark photoperiod, dissolved oxygen concentration from 5.3 to 6.8 mg/L, temperature 26.2 \(\pm\) 1.3 \({}^{\circ}\)C, pH 6.8-7.2, and ammonia and nitrite levels of 0.01-0.02 mg/L. Fish were fed feed of 5% body mass twice per day in the regular feeding time (9:00, 17:00). During cultivation, the health condition of fish in each group was observed and recorded every day. Dead fish was removed from the tank as soon as possible. At the end of the experiment, 10 fish were randomly selected from each tank for euthanasia with 3-aminobenzoic acid ethyl ester methane sulfonate (MS-222) (50 mg/L) (Adamas-beta, Shanghai, China). The middle part of intestinal specimens was dissected from anaest fish, placed in 1.5 mL sterile centrifuge tubes and stored in \(-\)80 \({}^{\circ}\)C for the microbial assay. The process was repeated five times with fifteen samples in each group.  
The other five intestinal specimens were removed, and fixed in paraformaldehyde fixing solution (Servicebio, Wuhan, China) for histopathology analyses. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was according to the National Institute of Health Guide for the Care and Use of Laboratory Animals of China. All experiments were approved by the Animal Care and Use Committee of South China Agricultural University (identification code: 20200127; date of approval: 20 May 2020). All surgery was performed under anesthesia with MS-222, and all efforts were made to minimize suffering.