Hgcl2

V. cholerae isolates were measured for in vitro susceptibility to ten antimicrobial agents (Oxoid, UK) according to the method described previously (Hu and Chen 2016 (link); Tang et al. 2014 (link)). Ten antimicrobial agents were AMP, CHL, KAN, RIF, SPT, STR, TET, TM, and sulfamethoxazole plus trimethoprim (SXT). Tolerance of V. cholerae isolates to eight heavy metals NiCl₂, CrCl₃, CdCl₂, PbCl₂, CuCl₂, ZnCl₂, MnCl₂, and HgCl₂ (Analytical Reagent, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was also determined according to the method described previously (Hu and Chen 2016 (link)). Escherichia coli strains ATCC25922 and K12 (Institute of Industrial Microbiology, Shanghai, China) were used as quality control strains in antibiotic and heavy metal resistance tests, respectively (Matyar 2012 (link); Song et al. 2013 (link)).

Cu(NO₃)₂, Na₂HPO₄, sodium ascorbate, boric acid, 1,4-dithiothreitol (DTT), and ethylenediaminetetraacetic acid disodium salt were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Tris(hydroxymethyl) aminomethane, NaCl, and MgCl₂ were bought from Sinopharm Group Co., Ltd. (Shanghai, China). HCl was bought from Hengyang Kaixin Chemical Reagent Co., Ltd. (Hengyang, China). SYBR Green I and DNA Marker were bought from Sangon Biotechnology Co., Ltd. (Shanghai, China). λ-Exo was obtained from New England Biolabs. Ltd. (Beijing, China). UO₂(NO₃)₂, Pb(NO₃)₂, HgCl₂, AgNO₃, CrCl₃, AlCl₃, FeCl₃, and KCl were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All the conventional chemical reagents were of analytical grade. The DNA oligonucleotides used were ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China) (Table S1).
The types of buffers used were as follows: buffer A (50 mM Na₂HPO₄ and 500 mM NaCl; pH 7.6), buffer B (50 mM Na₂HPO₄ and 50 mM NaCl; pH 7.6), buffer C (25 mM Tris-HCl, 50 mM NaCl, 2 mM MgCl₂, and 1 mM DTT; pH 9.4), and buffer D (50 mM Na₂HPO₄ and 1 mM EDTA; pH 7.6). Among these, buffer A was employed as the DNA stock solution (10.0 μmol·L⁻¹) of oligonucleotides, while the others were used as DNA reaction buffers. The solvent in each step was deionized water (DI water, 18 MΩ·cm) from a Milli-Q water purification system.

Crayfish were randomized into 4 groups (three replicates per group); in addition, twelve specimens per replicate. There was no replacement of water during the static exposure experiment. HgCl₂ (Sinopharm Chemical Reagent Company, Shanghai, China) at analytical grades was used. Previous study has indicated that 96 h LC50 concentration of Hg²⁺ for freshwater crayfish at 24 °C is 0.35 mg/L [56 (link)], in order to better evaluate the relationship between Hg concentration and toxicity; therefore, an appropriate Hg²⁺ exposure time of 96 h and temperature of 24 °C were chosen for this toxicity test. The Hg exposure concentration drew on research conducted with another heavy metal cadmium exposure on the crayfish [38 (link)], exposures of P. clarkii to Hg were respectively conducted at doses of 0 (Control group, Ctrl, 0 μg/L Hg²⁺), 1/40 LC50 (Low concentration group, Low, 8.75 μg/L Hg²⁺), 1/16 LC50 (Medium concentration group, Med, 21.875 μg/L Hg²⁺), and 1/8 LC50 (High concentration group, High, 43.75 μg/L Hg²⁺). Figure 1 illustrates the experimental procedure schematically. The doses of Hg²⁺ solutions were established by dissolving the desired amount of Hg²⁺ stock solution in dechlorinated tap water. The other exposure conditions were similar to those for the acclimatization described above. There was no crayfish death during the exposure experiments.

V. parahaemolyticus strains were measured for in vitro susceptibility to 10 antimicrobial agents and heavy metals according to the methods described previously (Song et al., 2013 (link)). The heavy metals used in this study included: NiCl₂, CrCl₃, CdCl₂, PbCl₂, CuCl₂, ZnCl₂, MnCl₂ and HgCl₂ (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China).