Copper-induced oxidative damage to the prophenoloxidase-activating system in the freshwater crayfish Procambarus clarkii

• Copper exposure induced carbonylation of haemolymph proteins in a dose-dependent manner.
• Protein carbonyls levels regulated enzyme activities of proPO, SP and PO.
• Carbonylation in hepatopancreas and haemolymph showed different effects on hemocyanin.
• Copper-catalyzed protein carbonylation suppressed the immune response in crayfish.
• This is the first report on protein carbonylation and subsequent decarbonylation in proPO-AS.

Previous studies have demonstrated copper-induced proteins damage in gill and hepatopancreas of the freshwater crayfish Procambarus clarkii, but little information is available about its effects on key component of the innate defense in haemolymph. In the present study, we evaluated the relationship between oxidative carbonylation and prophenoloxidase-activating system (proPO-AS) activity, by exposing P. clarkii to sub-lethal concentrations (1/50, 1/12, 1/6 and 1/3 of the 96 h LC50) Cu2+ up to 96 h. Six biomarkers of oxidative stress, i.e. reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), protein carbonyl (PC), malondialdehyde (MDA) and DNA-protein crosslinks (DPCs), and six indicators of immune status, i.e. total hemocyte counts (THCs), differential hemocyte counts (DHCs), hemocyanin (HC), prophenoloxidase (proPO), serine protease (SP) and phenoloxidase (PO), were determined in haemolymph. The results indicated that there was a significant increase (P < 0.05) in the levels of ROS, PC, MDA and DPCs accompanied by markedly decreased (P < 0.05) activities of proPO, SP, PO and HC in a dose and time dependent manner. The significant and positive correlations (P < 0.01) between ROS production and the formation of PC, MDA and DPCs were observed in crayfish at 96 h. There was a significant negative correlation (P < 0.01) between the levels of protein carbonyls and the activities of proPO and SP in hemocyte lysate supernatant and PO and HC in haemolymph. Carbonylated proteins may be recognized not merely as a specific signal in oxidative stress pathways but also as a “non-self” molecule in proPO-AS. In crayfish species, copper-catalyzed protein carbonylation may be one of the main mechanisms for immunity dysfunction in proPO-AS.