In vitro effects of noradrenaline on Akoya pearl oyster (Pinctada imbricata) haemocytes

Exposure to fluctuating environmental conditions in bivalve molluscs can lead to physiological stress and up-regulated production of stress-associated hormones, such as noradenaline (NA). Since environmental stressors have been found to have an immunosuppressive effect on Pinctada imbricata, we investigated the in vitro affects of NA exposure on their defensive haemocytes, focussing specifically on markers of apoptosis. Terminal dUTP nick-end (TUNEL) labelling was used to detect cells displaying DNA fragmentation within tissue exposed to NA. DNA fragmentation was most significant when haemocytes were exposed to 10.0 ng NA/μg protein relative to non-treated controls. Similarly, Annexin V-FITC staining, a marker of early apoptotic events, was evident in cells exposed to 5.0 and 10.0 ng NA/μg protein after 120 min (p < 0.05), and haemocyte adhesion to glass slides declined significantly when cells were exposed to 10.0 ng NA/μg protein (p < 0.05). A number of morphological and ultrastructural changes in NA-exposed haemocytes were also identified using transmission and scanning electron microscopy. These alterations included chromatin and cytoplasmic condensation, the formation of apoptotic bodies, vacuolisation and blebbing. In NA-treated cells, polymerisation of F-actin was observed around the periphery of the cytoplasm. All of these data suggest that NA induces apoptosis in P. imbricata haemocytes.