*2.2. Ammonia Nitrogen Stress*

A preliminary trial was conducted with six ammonia nitrogen concentrations: 0, 20, 40, 60, 80, and 100 mg·L<sup>−</sup>1. This allowed us to calculate the 96 h LC50 (medial lethal dose) [5,6]. There were 180 shrimp used in all. Thirty shrimp were housed in tanks each holding 3.5 L of saltwater with the desired amount of ammonia nitrogen. Every two hours during the preliminary experiment, mortality was noted. A linear regression equation was formulated to determine the median lethal (LC50-96 h) and safety concentrations (SC) [14,15]. It was found that the LC50 and SC concentrations were 50 and 5 mg·L−1, respectively. To control the level of ammonia nitrogen, clean seawater was mixed with ammonium chloride (NH4Cl). Three groups were employed in the experiment: pure seawater served as the control group, 50 mg·L−<sup>1</sup> represented a high level of ammonia nitrogen concentration, and 5 mg·L−<sup>1</sup> represented a low level of ammonia nitrogen concentration. Three parallel experiments were conducted in each experimental group. Every 2 h, shrimp survival in each experimental group was monitored and documented, and the dead shrimp were immediately removed from the bucket. After ammonia nitrogen stress, three shrimp were taken from each replicate at 0, 3, 6, 12, 24, 48, and 96 h. The shrimp's gills and intestinal tissues were rapidly removed and flash-frozen in liquid nitrogen.
