Biological effects of combined inorganic nitrogen stress on Penaeus vannamei under orthogonal design

The robustness of the results was enhanced by conducting two replicate experiments (the main difference being temperature fluctuation versus isothermal conditions). Phenotypic measurements were performed on water quality dynamics, growth performance, enzyme activity related to immune and osmotic regulation, histopathology, and apoptosis responses. Simultaneously, hepatopancreatic transcriptomic and hemolymphometabolomic analyses were combined to reveal the synergistic reprogramming of key pathways and their potential mechanistic connections. These results can provide experimental evidence and a biological explanatory framework for inorganic nitrogen risk identification, threshold control, and management strategy optimization in engineered aquaculture systems.