Manganese induced apoptosis in haematopoietic cells of Nephrops norvegicus (L.)

Manganese (Mn) is highly abundant as MnO2 in marine sediments. During hypoxia in bottom waters, the reduced bioavailable fraction of manganese, Mn2+, increases. Thereby, Norway lobster, Nephrops norvegicus, can experience concentrations up to 1000 times normoxic levels. A previous study has shown that exposure to a realistic concentration of 20 mg l−1 of Mn for 10 days reduced the number of circulating haemocytes in N. norvegicus significantly. Here we aimed to investigate if apoptosis contributes to the Mn-induced haemocytopenia, with the overall hypothesis that Mn induces apoptosis in a time and concentration dependent manner. N. norvegicus were exposed to Mn (0, 5, 10 and 20 mg l−1) for 5 and 10 days. After 5 days of exposure the total haemocyte counts were not affected. However, after 10 days there was a gradual decrease in cell numbers, reaching a reduction by 44% when the animals were exposed to 20 mg Mn l−1. Apoptosis in cells, released from the haematopoietic tissue, was investigated by using TUNEL assay, which detects specific DNA strand breaks. The fraction of apoptotic cells gradually increased from 2.5% in un-exposed lobsters to 15% in those exposed to 20 mg l−1 but there was no difference related to the exposure time. A gradual increase of apoptosis was further confirmed by electrophoretic DNA-ladder formation, however to a lower extent in lobsters exposed during 5 days. Cell viability, determined by metabolic activity and cell membrane integrity, was not reduced, indicating that apoptosis rather than necrosis caused reduced number of haemocytes. It was concluded that apoptosis seemed to increase already after 5 days of 5 mg l−1 of Mn-exposure, although exposure for 10 days was required before it was reflected in the haemocyte numbers. Reduced numbers of haemocytes may increase the prevalence for infections in N. norvegicus in their natural habitat.