Integrative Transcriptomic and Network Analysis of Hemocyte Volume Plasticity and Redox Regulation Under Osmotic Stress in Penaeus monodon

Osmotic stress affects ion transport and cell hydration, potentially disrupting redox homeostasis through altered proteostasis and mitochondrial metabolism. However, how immune hemocytes coordinate volume regulation with these stress-linked processes, particularly oxidative stress and antioxidant responses, remains unclear in crustaceans. This study integrated quantitative cytology, RNA sequencing, and network analysis to profile hemocyte volume plasticity in the euryhaline shrimp <i>Penaeus monodon</i> across a salinity gradient. Hemocytes were incubated for 24 h in hypoosmotic, isosmotic, and hyperosmotic media, with significant volume shifts observed while maintaining membrane integrity and morphology. The permeability of solutes (urea and sorbitol) suggested that volume adjustment is coupled with solute transport. Transcriptomic analyses identified key salinity-responsive pathways, including oxidative phosphorylation, MAPK signaling, ribosome biogenesis, and antioxidant defense mechanisms, underscoring the activation of redox-regulatory systems under osmotic stress. Weighted gene co-expression network analysis highlighted ribosomal proteins as central hubs in a salinity-responsive module, with qRT-PCR confirming the co-regulation of these hubs alongside representative osmoregulatory and antioxidant genes (<i>AQP4</i>, Na⁺/K⁺-<i>ATPase</i>, <i>HSP70</i>, <i>CHOP</i>, and antioxidant enzymes). These findings reveal how hemocyte volume dynamics are coupled to redox regulation, providing a mechanistic framework for understanding osmotic stress-redox coupling in crustacean immune cells.