Effects Of Cu²⁺ Stress On The Immune System Damage In Freshwater Crayfish Procambarus Clarkii

In recent years,with the development of industry and agriculture,water pollution problem become seriously.Copper?Cu?is common contaminant found in the aquatic environment,and its potential toxicological effects on aquatic organisms have received extensive attention.Even at 1.00 mg·L^-1 concentration,copper can suppress immune function and increase host susceptibility to pathogenic microorganisms of crustacean,which caused great economic losses to the aquaculture industries.The prophenoloxidase?proPO?activating system is an important non-self recognition system in invertebrates,which is closely related to the resistance to diseases.However,the toxicity mechanism of copper on pro-PO of crayfish is not clear.The current study assessed the sublethal toxicities induced by Cu in the fresh water crayfish Procambarus clarkii.The static test method of acute toxicity test was used.The nominal concentrations of Cu(1.0,3.0,5.0,10.0 mg·L^-1)corresponding to 1/20,1/7,1/4,1/2 of the 96 h LC₅₀(22.14 mg·L^-1).The study included samples from fresh water crayfish,haemolymph,gill and hepatopancreas.Each sample was analysed at three times points to determine the oxidative damage parameters such as reactive oxygen species?ROS?,superoxide dismutase?SOD?,glutaghione peroxidase?GSH-Px?,protein carbonyl?PCO?,DNA-protein crosslinking?DPC?and immune system indicates such as hemocyanin?Hc?,total hemocyte counts?THC?,prophenoloxidase?pro-PO?,phenoloxidase?PO?,serine proteinase?SP?.Moreover,Principal Component Analysis?PCA?and a correlation matrix were used to assess the interrelationships among the parameters used.The results suggested that:?1?ROS level in hemolymoh,gill and hepatopancreas was increased with the time extension and increasing dosage?P<0.05?;The SOD activity in gill decreased?P<0.05?.In serum,the SOD activity decreased in 5 mg·L^-1 treatment.The SOD activities in hepatopancreas increased and then decreased;Compare to the control,the GSH-Px activities in hemolymoh,gill and hepatopancreas was first increased and then decreased with the time extension and increasing dosage?P<0.05?;The PCO contents was increased in hemolymph,gill and hepatopancreas,but the 10.0 mg·L^-1 treatment didn't change significate in hemolymph;The DPC in gill and hepatopancreas increased with the time extension and increasing dosage.It's reached the peak in hemolymph treatment 3 mg·L^-1.All these results suggested Cu²⁺ is known to induce ROS and inhibit SOD and GSH-Px activities.The increasing PCO and DPC suggest higher ROS content could enhance the risk of DNA damage and protein carbonylation.Hemolymph is one of the main targets of ROS damage tissue.?2?Copper can suppress immune function of crustacean even at 1.0 mg·L^-1 treatment.Compared to the control,there is a significant decrease in THC and pro-PO activities in HLS of the crayfish?P<0.05?.Then,the pro-PO,SP and PO activities decreased in 10.0 mg·L^-1 treatment.It suggested Cu²⁺ may be involved in the regulation of proPO-AS.The hemocyte,intracellular and extracellular proteins,such as Hc,SP,PO and proPO,have different physiological effects.the PO activity increased along with the He content.In the present study,we suggest hemocyanin have the related functions that are mediated through phenoloxidase activity.Oxidative stress induced by Cu²⁺ possibly regulated proPO-AS through many different mechanisms.?3?According to PC A,3 principal components described the 9 original variables,explaining 78.40%of the total variance.The first principal component was associated with ROS,GPx,SOD,PO activities in serum and hemocyanin content.The second componentwas associated with DPC and PCO.The third component was associated with PO and serine proteinase in HLS.Based on this result,antioxidant indexes GSH-Px,SOD and immune indicator PO activity have a strong correlation with ROS content.PO and SP activities in HLS have a positive correlation.PO activity also has an positive correlation with He content.Then,we analysed the He and prophenoloxidase genes which download from genbank.The results suggested He and prophenoloxidase have the similar Cu²⁺ binding sites and hemocyanin C structural domain.It supported the opinion that He may have prophenoloxidase activity.In summary,Cu²⁺ have an inhibit effect on the pro-PO of crayfish Procambarus clarkii.The mechanism is:?1?Cu²⁺ induced oxidation and antioxidant imbalance.When the crayfish antioxidant defense system can not remove excess ROS,SOD activity and GSH-Px activity were suppressed.Protein and DNA is the most abundant biological macromolecules in cells and is the main target of ROS.The PCO content and DPC were increased and the proPO-AS activity or synthesis inhibited.?2?With the Cu²⁺ concentration increased and exposure time prolonged,THC?pro-PO?SP activities and He content inhibited.