Bif-1 upregulates autophagy to improve nitrite tolerance of Litopenaeus vannamei by interacts with Prohibitin-2

Nitrite toxicity is recognized as a pervasive environmental stressor in aquaculture systems, however, the molecular mechanisms by which Litopenaeus vannamei adapts to this stress remain incompletely elucidated. In this study, transcriptome sequencing is utilized to uncover an autophagy-mediated stress response. Differential expression analysis identifies 1729 differentially expressed genes, including 991 up-regulated and 738 down-regulated. KEGG enrichment analysis places autophagy and peroxisome among the top-ranked transport and catabolic pathways. A previously uncharacterized autophagy regulator, LvBif-1, is subsequently identified. The open reading frame spans 1083 bp and encodes a 360-amino-acid protein containing conserved N-BAR and SH3 domains. Sequence alignment reveals high identity with invertebrate orthologues. Tissue-specific expression profiling indicates high transcript abundance in hepatopancreas, heart, and muscle, and expression is markedly up-regulated under nitrite stress. Functional analyses demonstrate that RNAi-mediated silencing of LvBif-1 reduces levels of the autophagic markers LC3-II and Beclin-1, increases p62 accumulation, stabilizes mitochondrial membrane potential, elevates reactive oxygen species (ROS), and decreases hemocyte viability, ultimately exacerbating shrimp mortality. These phenotypes are partially rescued by the autophagy activator Rapamycin. Mechanistic investigations reveal that LvPHB2 interacts with the C-terminal region of LvBif-1. Upon nitrite stress, LvBif-1 translocates from the cytoplasm to the mitochondria. His pull-down assays further confirm that the interaction between LvBif-1 and prohibitin 2 (LvPHB2) is stress-inducible. Collectively, this study provides the first evidence that LvBif-1 alleviates nitrite toxicity by modulating autophagy, mitochondrial function, and the LvPHB2 interaction network. These findings deepen the molecular understanding of crustacean stress adaptation and offer a prospective target for breeding stress-resistant strains in aquaculture.