**2. Materials and Methods**

### *2.1. Chemicals, Characterization, and Strains Maintenance*

All of the chemicals used in this study were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Zinc chloride (ZnCl2) was used as a control to distinguish the effects of dissolved zinc ions (Zn2+). ZnO-NPs (<50 nm, assay grade, >97% purity) were sonicated and freshly prepared in 1000 mg/L stock solution before the assays. Characteristics of ZnO-NPs, including morphology and hydrodynamic diameter, have been previously documented [26]. The TEM image of ZnO-NPs used in the present study was shown in Figure S1. A previous study also reported the TEM image and X-ray diffraction (XRD) results of ZnO-NPs [33]. The concentrations of Zn released from ZnO-NPs in a Luria–Bertani broth (LB) medium during the 8 h *E. coli* exposure period were determined at 0, 4, 6, and 8 h at 37 ◦C. The initial concentration of the ZnO-NPs suspension prepared in the LB medium was 5 mg/L. The samples were filtered through Amicon Ultra-15 Ultracel-3 centrifuge tubes (3 kDa cutoff ≈ 0.9 nm, Millipore, Billerica, MA, USA) to remove the remaining ZnO-NPs, and the concentrations of Zn2+ in the aqueous phase were measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES, Spectro Ciros 120, Kleve, Germany).

*E. coli* OP50 and *C. elegans* strains, including wild-type N2 and transgenic EG1285 strain [*unc-47*p::GFP + *lin-15*(+)], were purchased from the *Caenorhabditis* Genetics Center (CGC, University of Minnesota, MN, USA). The freshly prepared overnight bacterial culture from a single colony was used for ZnO-NP exposure and dietary transfer assay. For maintaining the *C. elegans* strains, all of the worms were cultured at 20 ◦C on a nematode growth medium (NGM) agar plate covered with a bacterial lawn of *E. coli* OP50.

### *2.2. Measurement of Zn Concentration in E. coli OP50*

Saturated *E. coli* OP50 were freshly prepared and subcultured in the LB medium with various concentrations of ZnO-NPs for 8 h at 37 ◦C according to the methods of a previous study [20]. Subsequently, the bacterial culture was washed three times using deionized water by centrifugation (15 min at 3000× *g*). The *E. coli* OP50 pellet that was collected was digested using concentrated nitric acid (HNO3), and the Zn concentration was analyzed using ICP-AES.
