Two-Step Vibrio parahaemolyticus Challenge Reveals Transcriptional Reprogramming of Trained Immunity in Shrimp Hemocytes

Invertebrates rely exclusively on innate immunity but exhibit memory-like responses termed immune priming or trained immunity. In the commercially vital whiteleg shrimp (Litopenaeus vannamei), infection by Vibrio parahaemolyticus causes severe economic losses, yet the molecular networks driving secondary immune recall remain poorly understood. In this study, we established a two-step immune challenge model in L. vannamei using formaldehyde-inactivated V. parahaemolyticus and performed transcriptomic analysis on hemocytes to compare primary and secondary immune responses. Differentially expressed gene (DEG) screening and enrichment analyses (GO, KEGG, and GSEA) suggest that shrimp hemocytes undergo a broad and coordinated transcriptional reprogramming rather than uniform upregulation of immune genes. Transcriptomic data show potential associations between secondary immune priming and the modulation of cell fate processes: genes related to cell cycle progression (e.g., CDK1, CCNB3) and spindle assembly (e.g., MPS1) were significantly upregulated alongside apoptosis inhibition (CASP6 downregulation). Concurrently, metabolic remodeling was observed through the upregulation of lipid synthesis (SREBF1, FASN) and carbohydrate uptake pathways, potentially providing anabolic support for hemocyte growth and immune activation. Furthermore, the humoral effector responses appear to be strengthened, characterized by upregulated antimicrobial peptides (PEN, ALF) and the proPO melanization cascade (PPAF3, PPO3), whereas the expression of intracellular NLR was relatively suppressed, which might help mitigate excessive immune inflammation and immunopathological damage. Collectively, these transcriptomic findings identify a putative coordinated transcriptional signature of hemocyte recall responses in L. vannamei. This study expands our understanding of innate immune memory in invertebrates and provides candidate molecular markers for further study in disease-resistant breeding research in shrimp aquaculture.