Put dihydrochloride

Pearl oysters (∼1.5 years old) were obtained from Houhong, Zhanjiang, Guangdong, China. The reagent was injected into the adductor muscle via a 100 μl microsyringe. The challenged group was injected with 100 μl of 0.5 mg/ml LPS (Sigma-Aldrich, St. Louis, MO, United States). The control group was injected with 100 μl of 1 × phosphate-buffered saline (1 × PBS) in the same way. In the Put group, 1 h before LPS injection, 100 μl of 1 mM Put dihydrochloride (Sigma-Aldrich, St. Louis, MO, United States) was injected. The pearl oysters injected with 100 μl of the 1 × PBS solution were used as the control group before LPS stimulation (i.e., LPS group). After LPS stimulation, the pearl oysters were maintained at (25 ± 1)^°C in cistern with filtered seawater. Hemolymph was then extracted from eight pearl oysters in each group by using a 2 ml syringe at 6, 12, 24, and 48 h after LPS stimulation. Hemocytes and serum were separated from the hemolymph via centrifugation at 3,000 r/min for 5 min at 4°C. The collected hemocytes were resuspended in the TRIzol reagent (Invitrogen, Carlsbad, CA, United States). Both serum and hemocytes were quick-frozen in liquid nitrogen and stored at −80°C until use.