Effects Of Cu²⁺ On Several Isoenzymes In Macrobrachium Rosenbergii

Macrobrachium rosenbergii with delicious flavor and high nutrition was popular with more and more people and bred in continually enlarging area. Unfortunately, the shrimp farming business was intimidated potentially by the abolished water from our daily life and factories, especially by the frequently using of copper sulfate for sterilizing the pools and all that above caused the increasing of Cu²⁺ in farming water. Copper was an essential nutrient element in crustacean, which played an important part in enzymes related with development and growth. But excess Cu²⁺ could be harmful. The concentration of Cu²⁺ should be less than 0.01mg/L, pointed out by Chinese fishery ecology condition reports in 2003. While in some places, such as Hangzhou Bay, the Yangtze River exit, it had been constantly increasing even over than 0.07mg/L. So it was meaningful not only for exploring the effects of Cu²⁺ on the shrimp's growth mechanism but also for searching healthy way to breed crustacean. As a biochemical measurement, isoenzyme had been widely used to analyze relationships between species, biological system classification and gene regulation etc. Many reports about the heavy metals toxicity on crustacean had been found recently, but the effects of Cu²⁺ on isoenzyme in Macrobrachium rosenbergii hadn't been reported frequently yet. Single gradient factor experiment was applied in this research and based on concentration of Cu²⁺ contained in polluted water and the way of sub-acute toxic experiment, we set a concentration gradient, 0.01mg/L, 0.08mg/L, 0.64mg/L and 5.12mg/L. In order to discuss the influence and stress of different concentration of Cu²⁺ on Macrobrachium rosenbergii and try to give some suggestions for properly breeding, through three main methods (enzyme activity test, isoenzyme electrophoresis pattern analysis and enzyme cytochemical localization by electronic microscope ), we researched the changes of several isoenzymes(including exterase (EST), phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and peroxidase (POD)) in Macrobrachium rosenbergii treated by different concentrations of Cu²⁺ after one week. 1. Effects of Cu²⁺ on EST, POD and SOD enzyme activity in five tissues of Macrobrachium rosenbergiiThe changes of three enzyme activity (EST, POD and SOD) in Macrobrachium rosenbergii treated by different concentrations of Cu²⁺, were analyzed by spectrophotometer and the statistically significant differences were tested by SPSS software.Changes of EST enzyme activity: the changes in eye, muscle and heart were slight and no statistically significant differences were found. In gill, the EST activity enhanced by low concentrations of Cu²⁺ and got the maximum when Cu²⁺ was 0.64mg/L. Later, 5.12mg/L Cu²⁺ let it drop and all the Cu²⁺ treated groups had statistically significant differences compared with the control (P<0.05). In liver, totally speaking, EST activity firstly increased and then decreased. It got climax when Cu²⁺ was 0.01mg/L and except a bit increase was found when Cu²⁺ was 0.64mg/L, the rest all decreased along with the increasing of Cu²⁺. There were statistically significant differences between Cu²⁺ treated groups and the control (P<0.05).Changes of POD enzyme activity: the changes in eye, muscle and heart were slight and no statistically significant differences were found. POD activity in gill enhanced by low concentrations of Cu²⁺ and inhibited by 5.12mg/L Cu²⁺. It got climax when Cu²⁺ was 0.64mg/L and statistically significant differences were found between the treated groups and the control (P<0.05). In liver, POD activity fluctuated a bit. Firstly, it increased and got climax when Cu²⁺ was 0.01mg/L. After that, it droped except a bit increase when Cu²⁺ was 0.64mg/L. Statistically significant differences were found between the treated groups and the control (P<0.05).Changes of SOD enzyme activity: Both of the five selected tissues's SOD enzyme activities were dramatically enhanced by low concentrations of Cu²⁺. It got climax in eye and heart when Cu²⁺ was 0.08mg/L and in the rest three tissues, it got highest when Cu²⁺ was 0.64mg/L. The highest concentration (5.12mg/L) of Cu²⁺ inhibited the SOD activity in all of the five tissues. More statistically significant differences were found in gill, liver and heart between the treated groups and the control.All in all, along with the increase of Cu²⁺, EST, POD and SOD activity both ascended firstly and descended at last as a whole which suggested a dose---effection relation between the concentration of Cu²⁺ and enzyme activity. This kind of relation was especially obvious in gill and liver. So maybe it was possible to know the stress of Cu²⁺ on Macrobrachium rosenbergii by analysing the changes of several related enzyme activity in gill and heart.2. Influences of Cu²⁺ on Isoenzyme electrophoresis patterns of EST, POD and SOD in five tissues from Macrobrachium rosenbergiiVertical polyacrylamide gel electrophoresis (PAGE) and biochemical staining were used to analyze five isozymes including EST, POD and SOD in the eye, liver, heart, gill and muscle of Macrobrachium rosenbergii treated by different concentrations of Cu²⁺ for one week. The specific tissue expression was found and the result was introduced in details as follow:Changes of EST electrophoretogram: EST was expressed especially strongly with more enzyme bands in liver and gill and no band was missed or generated with the increase of Cu²⁺ in all of the five tissues. No obvious changes were found in electrophoretograms of eyes, heart and muscle. In gill, all the six enzyme bands changed regularly. They both got increasingly darker with the increase of Cu²⁺ until 5.12mg/L Cu²⁺ lighted them a bit. In liver, EST got the strongest expression and there were five bands altogether. Among the five bands, three bands(Rf=0.078,0.703,0.859) changed obviously and followed the changes of EST enzyme activity. The other two bands(Rf=0.391,0.469) gave no special changes.Changes of POD electrophoretogram: There were less enzyme bands and slight changes of POD electrophoretograms in eyes, heart and muscle. In gill, the width and darkness of enzyme bands both ascended and got the peak when Cu²⁺ was 0.64mg/L. After then, enzyme bands narrowed and the color lighted a bit. POD was stongly expressed and there were two bands (Rf=0.078,0.203) altogether in liver. One band ( Rf =0.078) fluctuated a bit and the other one (Rf=0.203) broadened and became darker when the concentration of Cu²⁺ got higher. It was not got narrowed and lighted until the Cu²⁺ was 5.12mg/L.Changes of SOD electrophoretogram: There were less enzyme bands and slight changes of SOD electrophoretograms in eyes, heart and muscle along with the increase of Cu²⁺. In gill, there were three bands (Rf=0.157,0.393,0.881) and among the three, two bands (Rf =0.157,0.881) got broadened as the increase of Cu²⁺. While 5.12mg/L Cu²⁺ had them narrowed. The Darkness of the former one (Rf =0.157) changed slightly while the later one's darkness increased and got darkest when Cu²⁺ was 0.64mg/L. A new band ( Rf =0.393) was generated when Cu²⁺ was 0.08mg/L and 0.64mg/L. In liver, there were three bands(Rf=0.157,0.393,0.674) and among the three, Rf =0.157 band got broadened along with the increase of Cu²⁺ while no obvious changes of darkness was found. Rf=0674 band got broadened and darker until 5.12mg/L Cu²⁺ had it narrowed and lighted. A new band ( Rf =0.393) was generated when Cu²⁺ was 0.01mg/L, 0.08mg/L and 0.64mg/L.In a word, along with the increase of Cu²⁺, width and colorness of the three kinds of isoenzymes bands both changed more or less and it was dramatically obvious in gill and liver. What's more, a new band was aroused and missed in gill and liver electrophoretograms. So this phenomenon may suggest gill and liver were much more sensitive to the changes of Cu²⁺ contained in the farming water. The reason to explain this phenomenon might be that gill was prawn's main breath organ and contacted the heavy metal ions directly and liver was the main cumulation place of heavy metals. So in our view, to research the influences of Cu²⁺ on Macrobrachium rosenbergii's normal physiological reaction, it was feasible by analyzing the changes of some isoenzymes electrophoretograms in gill and liver.3. Influences of Cu²⁺ of Cytochemical Location of some Isozymes (POD, SOD, ACP and AKP) in Macrobrachium rosenbergiiEnzyme cytochemical technique of electronic microscope was used in this article to research the changes of cell ultrastructure and enzyme location of POD, SOD, ACP and AKP between the Macrobrachium rosenbergii treated by 5.12mg/L Cu²⁺ for one week and the healthy ones. The results were introduced in details as follows:Changes in cell ultrastructure: In healthy Macrobrachium rosenbergii, we found that, round pigment granules, normal karyon, mitochondrium etc in eye cell; normal mitochondrium, karyon, lots of perfect and smooth lamellas in gill; assorted cell ultrastructures like mitochondrium, lipid droplets, microvilli, lysosome and peroxisome etc in liver cell and most of them were in good condition; in heart cell, lots of mitochondriums which were full of mitochondrial cristaes and many myocardial fiber were arranged orderly; in muscle cell, there were many muscle fiber arranged orderly and lots of clear normal karyon, mitochondrium etc. While in Macrobrachium rosenbergii treated by 5.12mg/L Cu²⁺ after one week, we found that, karyon pyknosis, many pigment granules were destroyed and cytoplasm was turbidity in eye cell; mitochondrium bulged, less mitochondrial cristaes, illegible cell ultrastructures, more myelin figures, karyon pyknosis were found in gill cell; in liver cell, karyon pyknosis, destroyed lipid droplets, blurred cytoplasm, abnormal expansion of endoplasmic reticulum and half-baked cellular membrane were found; in heart cell, shrinked karyon arounded by several vesicles , sarcoplasmic reticulum expansion, less mitochondrium, mitochondrium bulged, mitochondrial cristaes decreased and shrinked into one corner, myocardial fiber ruptured a bit; in muscle cell, more myelin figures, expansion of sarcoplasmic reticulum and ruptured muscle fibers were found. The result mentioned above suggested that 5.12mg/L Cu²⁺ could destroy cells in the five tissues more or less and especially obviously in gill, liver and heart. Maybe the function of the different tissues and the cumulation of Cu²⁺ could explain this phenomenon.Changes of enzyme locations: In healthy Macrobrachium rosenbergii, POD activities was mainly deposited in peroxisome, nucleus and around lipid droplets in liver cell; in Cu²⁺ treated Macrobrachium rosenbergii, peroxisome decreased and POD positive granules in nucleus disappeared. Positive granules around lipid droplets became less. POD got less expression which supported the results in enzyme activity test and isoenzyme electrophoretograms analysis. SOD activities in healthy Macrobrachium rosenbergii was mainly deposited in nucleus, the surrounding of lipid droplets and mitochondrial membrance etc. While in Cu²⁺ treated Macrobrachium rosenbergii, SOD positive granules became less around lipid droplets and mitochondrial membrance and disappeared in nucleus. SOD expression was inhibited obviously in lines with the results of SOD enzyme activity test and SOD isoenzyme electrophoretogram. The two isoenzymes related with immune function both were inhibited, which suggested that high concentration of Cu²⁺(5.12mg/L) destroyed body immune function and made the body more vulnerable to bacterium and virus infection.In healthy Macrobrachium rosenbergii, ACP activities were mainly deposited in nucleus, lysosome and sarcoplasmic reticulum. Being treated by Cu²⁺ after one week, more ACP positive granules were found in nucleus, lysosome, sarcoplasmic reticulum and cytoplasm which suggested ACP expression got abnormally enhanced. In healthy Macrobrachium rosenbergii, AKP activities were mainly deposited around nuclear membrane, cellular membrane, lipid droplets and sarcoplasmic reticulum. In Macrobrachium rosenbergii treated by 5.12mg/L Cu²⁺ for one week, more AKP positive granules were found around nuclear membrane, cellular membrane, lipid droplets, sarcoplasmic reticulum and other abnormal membranous structures in cytoplasm which suggested AKP expression got abnormally enhanced. The increased expression of ACP (the symbol enzyme of lysosome) may suggest that there were large amount of malfunctional organelles which stimulated the body to produce more lysosomes containing ACP to digest them. AKP, with a close relation to membrane, got enhanced may suggest the synthesis of membrane enhanced. This may caused by the need of more membrane to produce secretory vesicles to transport the giblets left by lysosomes.In a word, more or less changes could be found by enzyme activty test, isoenzyme electrophoretogram analysis or enzyme cytochemical location analysis. The changes were much more obvious in gill and liver. Maybe it was because gill was prawn's main breath organ and contacted the heavy metal irons directly and liver was the main cumulation place of heavy metals. So the two tissues (gill and liver) were much more sensitive to the changes of Cu²⁺ contained in the farming water. Therefore, it was possible to research the influences of Cu²⁺ on Macrobrachium rosenbergii by analyzing the changes of several isoenzymes in the two tissues mentioned above. But the factors causing the changes of isoenzyme were complicated and there were other kinds of heavy metals except copper. So much more efforts should be put into this way to analyze the influences of Cu²⁺ on Macrobrachium rosenbergii.