Flow cytometic analysis of Penaeus monodon haemocyte responses to poly I:C.

This study was aimed at investigating the cellular responses of Penaeus monodon haemocytes to poly I:C stimulation using flow cytometric assay. Total haemocyte count (THC), percentages of different haemocyte subpopulations [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)], non-specific esterase activity (EA), total reactive oxygen species/reactive nitrogen species (ROS/RNS) production, nitric oxide (NO) production, apoptotic haemocyte ratio and plasmic phenoloxidase (PO) activity were determined in poly I:C-injected shrimp. Results showed that poly I:C at a low dose (5 μg shrimp) caused obvious increases in THC, GC proportion, ROS/RNS production and NO production, but had no significant effect on EA, apoptosis and PO activity. In the early stage of poly I:C injection at a high dose (20 μg shrimp), THC and GC proportion improvements could also be observed, suggesting that GC might be induced to release from hemocytopoietic or other tissues to participate in immune response, and this subpopulation might be the main cell type involved in the cellular defence against virus. In the later period, proportions of both GC and SGC reduced paralleled by THC reduction, indicating that depletion of GC and SGC was mainly contributed to the reduced count of circulating haemocyte. Obvious increases in ROS/RNS production and NO production were induced in haemocyte of shrimp under a high dose of poly I:C stimulation, but only slight rise of EA and suppression of PO activity could be observed in poly I:C-stimulated shrimp, suggesting that ROS/RNS-dependent system was vital in the immune defence of shrimp against virus. On the other hand, increase of apoptotic haemocyte ratio and THC reduction were presented after the drastic increases of ROS/RNS and NO productions, implying that the stimulated ROS/RNS might be excess and harmful, and was the major factor for the haemocyte apoptosis and depletion. THC recovered after 48 h injection, while haemocyte apoptosis also returned to the control level, suggesting that apoptosis might be contributed to eliminate damaged, weak or infected haemocytes to renew the circulating haemocytes, and it could be considered as an important defending strategy against virus.