Cadmium Toxicity On Hemocytes Of The Freshwater Crab Sinopotamon Henanense And The Modulation Of Lipopolysaccharides

Cadmium(Cd)is a toxic heavy metal pollutant and is known to exert adverse effects in aquatic organisms.In order to study the toxity of sublethal Cd on hemocytes of the freshwater crab(Sinopotamon henanense)and the modulation of lipopolysaccharides(LPS),three concentrations of Cd(0.725,1.450 and 2.900 mg/L)and a control group were set.After Cd treatments for 7,14 and 21 d,the individuals were divided into two equal parts.Cd toxity on the hemocytes were determined using one part,the other part of individuals were injected with LPS(10?g/mL)for 24 h in order to detect the modulation of LPS on Cd toxity.After treatments,the hemocytes were collected from each individul.Firstly,changes of hemocytes ultrastructure including cell membrane,nucleus and organelles(mitochondria,lysosome and endoplasmic reticulum)were observed by transmission electron microscopy(TEM)technology,morphology damages in hemocytes caused by Cd were primarily studied.Secondly,Cd accumulation in hemocytes was measured by the atomic absorption spectrophotometer.The total number of hemocytes(THC)was determined using a hemocytometer.The reactive oxygen speicies(ROS)level,activities of acid phosphatase(ACP),alkaline phosphatase(AKP)and lysozyme(LSZ)were measured by the spectrophotometer method.In addition,the stability of lysosomalmembrane was detected using the method of neutral red staining.Cd accumulation in hemocytes led to ROS level increasement,resulting in oxidative damage.The changes of physiological and biochemical functions caused by ROS were further studied,the effects of LPS on the Cd toxicity were researched as well.Thirdly,the activities of total antioxidant capcity(T-AOC),antioxidant enzymes including: superoxide dismutase(SOD),catalase(CAT),glutathione peroxidas(GPx)and peroxidase(POD),malondialdehyde(MDA)level were measured by the spectrophotometer method.Protein carbonyl derivates(PCO)contents were also determined by the method of 2,4-DNPH chromatometry,and DNA-protein crosslink(DPC)levels were measured by using the method of “KCL-SDS” precipitation as well.This part of study was aiming to illustrate the effects of Cd on the oxidative damage and the regulation of LPS on these oxidative damage induced by Cd.Finally,the expression levels of immune-related genes including prophenoloxidase(proPO),lysozyme(LSZ)and metallothionein(MT)were detected by the real-time fluorescence quantitative PCR(qPCR)method.Also,the PO activity in hemocytes was measured by the spectrophotometer method.Stress responses mechanism of Cd in crab(S.henanense)and the modulation of LPS were deepen explored on the molecular levels.The results showed that:1.Effects of Cd on ultrastructure of hemocytes in the freshwatercrab S.henanenseThe hemocytes of crabs were classified into three types including large granular cells,semi-granular cells and hyaline cells.Ultrastructure of hemocytes after Cd treatment were damaged to a certain degree.Cd caused granular cells degranulation and the decrease in the number of particles in the cytoplasm,cell membrane and the nucleus became irregular,chromatin condensed,mitochondrial cristae fractured,cavity even disappeared.Also,Cd led to rough endoplasmic reticulum(rER)cavity expansion and the attached ribosomes on rER fell off,lysosome membrane blurred and a number of secondary or tertiarys appeared.More importantly,the results also showed that higher concentrations of Cd(2.900 mg/L)exposure duration from 7 d to 21 d,the ultrastructure of hemocytes were damaged more seriously than those of the lower concentration group(0.725 mg/L).2.Immune-associated parameters responses to Cd in the freshwater crab S.henanense and the modulation of LPSCd exposure elicited a significant accumulation in hemocytes of crab,a decrease in THC,and the production of ROS in the hemocytes of crabs.Treatment with Cd further decreased the stability of lysosomal membranes in hemocytes and induced substantial changes of immune-related parameters including ACP and AKP in hemocytes.Compared with Cd treatment alone,THC and ROS production in crabsexposing to Cd followed LPS injection were decreased in some extent.The stability of lysosomal membranes also decreased after LPS injection.The activities of ACP and AKP increased as well.However,the activity of LSZ varied weakly through out the whole treatment period.3.Antioxidative enzymes and oxidative damages in the hemocytes of the freshwater crab S.henanense exposed to Cd and the modulation of LPST-AOC was significantly inhibited by Cd and LPS enhanced the activity of T-AOC in hemocytes of the crab.In addition,treatment with Cd also changed the activities of antioxidant enzymes including SOD,CAT,GPx,and POD,in a trend of “up-regulate firstly then drop down”along with Cd concentration increasing and exposure time prolonging.Levels of PCO,DPC and MDA obviously increased in hemocytes,compared with the control group.Compared with Cd treatment group alone,LPS injection could modulate the changes of these antioxidant enzymes activities and oxidative parameters to some extent.4.Immune-related genes responses in the hemocytes of the freshwater crab S.henanense exposed to Cd and the modulation of LPSThe expression level of proPO mRNA was down-regulated through Cd exposing duration.Compared with the relative Cd group alone,LPS injection followed Cd treatment,changes of proPO mRNA expressionlevel were not obvious.Also,PO activity increased,and peaked at 14 d(0.725 mg/L,Cd),while it's activity decreased with Cd concentration increasing or expsure time prolonging.PO activity showed down-regulation in whole after LPS injection,compared with Cd group alone.Moreover,the expression levels of LSZ and MT mRNA were up-regulated significantly,showing dose-effect and time-dose-effects respectively.In addition,compared with Cd treatment alone,LSZ and MT mRNA expression levels in the hemocytes of crab were down-regulated after LPS injection.These results indicated that:1.The ultrastructure of hemocytes were changed when S.henanense were exposed to Cd,this may affect the process of synthestis and secretion of immune related factors,depressed normal immune function of crabs.2.Cd accumulation in hemocytes of crab caused higher level of ROS,resulting in damage of lysosome,releasing lysosomal enzymes.LPS could down-regulate ROS level and may improve the immunity in some extent in crustaceans.3.Cd was deleterious to the hemocytes of S.henanense,and the oxidative damages were caused by Cd through depressing antioxidant enzymes activities,leading to the decrease of immunity of the hemocytes or individuals finally.The oxidative damages caused by Cd exposurecould be alleviated by LPS,suggesting its use in aquaculture as a potential immunostimulant for crustaceans.4.Immune-related genes were affected obviously by Cd and modulation effects of LPS on these parameters were various in the crab.The results could provide a scientific basis for exploring the mechanism of Cd stress on the immune system of crustaceans.Meanwhile,the results could provide basic knowledge to improve the quality of aquatic products using LPS as a immunopotentiator in aquaculture.In conclusion,this study is of great significance on researching the disease defense in crustaceans.