| Rotifer is one of the important groups of planktonic animal.The smallest of rotifer is only about 50?m,the largest up to 2mm,whose life cycle is very short,between 5 to 15 days.The figures of rotifersare various.mostly oblong.And its body is divided into three parts:head,torso and legs.The body is not segmented,with false cavity.It has typical features like corona with cilia,specialized mastax and excretory system of protonephridia.There are about 2200 varities of rotifers discovered to date,belonging to Seisonidea,Bdelloideaand Monogononta.in the process of animal breeding,in order to cope with the harsh environment,some embryo would be wrapped with a layer of coating film outside.During this time,the animal embryo is in the period of dormancy to wait for the right conditions for development.This animal embryo is called the resting eggs.When rotifers meet with adverse environments,such as sudden drop in temperature,heavy metals and other stimulations,resting eggs will be generated in response to the adverse environment.The formation of resting eggs is one of the most remarkable characteristics of Seisonidea rotifer life cycle.The resting eggs are not only a self-protection mechanism to cope with the environment,but also play an important role in the human production.In the early period of larval shrimp hatched,the lack of open bait is difficult problem for breeders,and the uncertainty of breeding rotifers adds cost,so it is very urgent to develop rotifer resting eggs which are convenient to conservation and transportation.There is not much research on low concentration of copper ion on Brcachionus calyciflorus,so this study will focus on the low concentration of copper ion on Brachionus calyciforus ecological index and resting eggs,and the enzyme activity and mRNA expression.1.Effects of low concentration of copper ion on rotifer resting eggsWith the increase of copper ion concentration,the resting egg production of rotifer Brachionus calyciflorus also increased.At 0?g/L and 2.5?g/L copper ion concentration,the changes of resting egg production are minor.At 2.5?g/L copper ion concentration,the ecological indicators did not change.When at 5?g/L,rotifer restingegg production changed obviously,after the 8h the resting eggs were generated,earlier than at 0?g/L and 2.5?g/L.With the increase of copper ion concentration,the number of resting eggs showed slight fluctuation,not significant overall change.When treated at5?g/Lcopper ion concentration,rotifer resting eggs number was increased.Along with the increase of the concentration of copper ion,the over-breeding of rotifer resting eggs also came in advance.At 12.5?g/L concentration of rotifers have finished generating all the resting eggs.After 24h the amount will stop increasing.It indicates that when the copper ion is higher in concentration the rotifer will die after generating large amount of resting eggs.2.Study of SOD and CAT enzyme activity changes of the low concentration of copper ion stressIn this experiment at a certain range of concentration,different concentrations of copper ions stress can cause the Brachionus calyciflorus cells SOD and CAT activity increasingsignificantly(P<0.05)compared with the control group.CAT showed a downward trend after reaching a peak,and SOD is maintained at a high level,but decreased after 24h.The stress of copper ion increased the ROS ion and H2O2 levels of rotifers' culture.The stress signaling pathways including the JAK/STAT signal pathway,stimulate the cells and activate the antioxidant defense system SOD and CAT enzyme on behalf in the operation.The transcription and translation levels rise,in response to heavy metal stress environment,which reduce the production of rotifer cell damage effect.At last the performance increased by SOD and CAT enzyme activity.JAK/STAT signal pathway played an important role in promoting oxidative stress injury.Neutrophil accumulation and cell apoptosis,including oxidative stress damage can be determined by measuring SOD activity to determine proof.Fang Fang research shows that,the peroxidase activated receptor can protect the body by JAK/STAT pathway.Heavy metal stimulation can generate H2O2 organism.In the H2O2 induction of adaptive protection response through a variety of molecular mechanisms of anti apoptosis,involves the activation of several signaling pathways.The experiment with the two enzyme activity reached the peak after the decline.The main reason is when the heavy metal ion concentration is too high,the copper ion may be combined with the protein molecule with carboxyl or amido acid,leading tothe inhibition of enzyme system activity is inhibited.The cell can't function properly,resulting in the death of rotifers.3.Study on the effect of low concentration of copper ion on the expression of each geneThe experiment showed that the design of the 5 experimental groups,the concentrations of 2.5?g/L,5 ?g/L,7.5?g/L,10?g/L and 12.5?g/L,with a control group of 0?g/L.The time point of design was Oh,0.5 h,1 h,2 h,3 h,6 h,12 h and 24 h.The amounts of each gene expression at different time points were measured under different consequent.Bc CAT by copper ion influence,at 2.5?g/L the expression ofthegenes is higher than other groups.Bc CuZnSOD by copper ion had little effect on each time point,in addition to 12.5?g/L-6h.Bc MnSOD at 2.5?g/L and 5?g/L concentration,has the strongest gene expression at 1h,the amount of gene expression reached the peak,then decreased.The peak of hsp-40 appeared at 7.5?g/L-3h.With increase of the concentration,expression of hsp60 gene significantly increased in quantity and hsp70 also showed changes similar to hsp60.hsp90 is higher than hsp70,but also appeared a significant increase.4.Effects of vitamin E on Brachionus calyciflorusVitamin E can effectively reduce the rotifer resting egg production,and the result effects on enzyme activity.With the increase of vitamin E concentration decreased gradually,resting egg production of rotifer reduced and generation time also delayed.Prolongeing the asexual era of rotifer resting eggs plays an important role in the production.By adding zinc,production of rotifer resting eggs increased with the zinc ion concentration and then decreased.Presumably zinc ion concentration causedhigh adult mortality,the two did not significantly change.In the vitamin E stimulation,the expression of various genes also changed.The change of hsp70 is not obvious,other genes have corresponding changes.5.Effects of low concentration of copper ion onBrachionus CalyciflorusBrachionus calyciflorus rotifer is effected at low concentrations of copper ion concentration,not only ecological index changedat 2.5?g/L,but although no changes in the ecological index of rotifer.However,we can find that the activity and mRNA expression had changed a lot compared with the control group through the experiment.The copper cannot stimulate Brachionus calyciflorus body to produce stress mechanism,but the active of CAT,SOD enzyme and gene expression increased.Copper ion toxicity resistance increased,too.The ecological index does not reflect the changes of various indexes of the body,especially the enzyme,gene etc.That needs to be further verified by experiments. |
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