Hemocyanin is associated with GAPDH-related glycolytic metabolism during anti-Vibrio responses in Penaeus vannamei.

Immunometabolic reprogramming is a hallmark of host defense. Recent studies have shown that hemocyanin regulates the tricarboxylic acid (TCA) cycle in shrimp to enhance immune competence, yet its role in glycolytic regulation remains unclear. Here, we show that RNA interference-mediated silencing of hemocyanin in Penaeus vannamei markedly reduces the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme. Hemocyanin knockdown also decreases GAPDH protein abundance and enzymatic activity, accompanied by reduced levels of 1,3-bisphosphoglycerate and NADH. Although no direct physical interaction between hemocyanin and GAPDH was detected, co-expression of PvHMC was associated with increased total GAPDH enzymatic activity in the heterologous HEK-293T system, suggesting an indirect functional association. Upon pathogen challenge, both hemocyanin and GAPDH responded dynamically, and exogenous NADH supplementation partially restored the expression of representative glycolytic and TCA-cycle enzymes in hemocyanin-silenced shrimp. Importantly, NADH supplementation reduced Vibrio parahaemolyticus burden and improved host survival after infection. Together, these findings support a model linking hemocyanin with GAPDH-related glycolytic metabolism and NADH homeostasis.