Immune parameter changes of hemocytes in green-lipped mussel Perna viridis exposure to hypoxia and hyposalinity

Marine hypoxia owing to nutrient enrichment of water bodies by anthropogenic activities has become a global problem for several decades. Another marine environmental factor, salinity, plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where hypoxia frequently occurs. The present study evaluated the interactive effects of hypoxia and salinity on immune functions of hemocytes in the green-lipped mussel Perna viridis, which widely distributes in estuarine and coastal waters of the Indo-Pacific region. Mussels were subjected to six combinations of oxygen levels (hypoxia: 1.5mg O₂ l⁻¹, normoxia: 6.0mg O₂ l⁻¹) and salinities (15‰, 22‰ and 30‰) for 168h. Mussel hemocytes were sampled after 24h, 48h, 96h and 168h exposure, for analysis of immune functions including hemocyte mortality, phagocytosis, esterase, reactive oxygen species (ROS) production, lysosomal content and total hemocyte counts using flow cytometric methods. Thereafter, all treatments were recovered to normoxic condition (6.0mg O₂ l⁻¹) and salinity 30‰ for 24h to study the recovery of immune functions from the combined hypoxic and hyposaline stresses. Interactive effects of dissolved oxygen (DO) and salinity on all immune parameters were observed during the experiment. Generally, hemocyte mortality was higher at low salinity than that at high salinity levels; higher hemocyte mortality was also noted when mussels were exposed to hypoxia compared with normoxia. Phagocytosis at low salinities and hypoxia was decreased compared to normal DO and salinity treatment, respectively. The activity of esterase and ROS production at low salinity and hypoxia were reduced, respectively. Lysosomal content and total hemocyte counts were lower at low salinity and hypoxia compared to other treatments, respectively. After 24h recovery, hypoxia and salinity effects were still significant among previous treatments, indicating immune functions of P. viridis could not recover from combined stresses in a short time.