Studies On Thetoxical Effects Of Chlorpyrifos On The Cultured Shrimp, Litopenaeus Vannamei

To evaluate the toxicity of chlorpyrifos on the cultured shrimp Litopenaeus vannamei,the acute toxic tests of chlorpyrifos on juvenile shrimp (4.88±0.11g) and adult shrimp(10.27±0.41g) were carried out. The results showed that the death time was furthershorten and died earlier with the concentration of chlorpyrifos increasing;For juvenileshrimp, the24hLC50,48hLC50,72hLC50and96hLC50of chlorpyrifos were3.948μg/L,2.695μg/L,1.320μg/L and0.758μg/L, respectively, andthe safe concentrationwas0.0758μg/L; For adult shrimp,the24hLC50,48hLC50,72hLC50and96hLC50ofchlorpyrifos were5.743μg/L,3.688μg/L,2.437μg/L and1.951μg/L, respectively,and thesafe concentration was0.195μg/L. According to the pesticide toxicity grading criteria,toxicity level of chlorpyrifos on the cultured shrimp Litopenaeus vannamei was highlytoxic. Besides, compared to the juvenile shrimp, the adult shrimp was more sensitive tochlorpyrifos.In order to evaluate the hazard of chlorpyrifos on the cultured shrimp Litopenaeusvannamei further，the lethal effect on the shrimp infected White Spot Syndrome Virus(WSSV) under the chlorpyrifos stress wasanalyzed, while the WSSV copies in the gilland acetylcholinesterase(AChE) acticity in the muscle tissue were also tested. Based onthe results of acute toxicity tests, the stress concentration of chlorpyrifos of0.2μg/L wasdetermined. After the shrimp were exposed to0.2μg/L chlorpyrifos for4days, theywere injected WSSV. The resultsdemonstrated that the cumulate mortalities ofchlorpyrifos–WSSV treatment (83.33%±4.7%) were significantly higher than that ofethanol–WSSV group (40%±0.9%). In addition, the amount of WSSV were alsodetermined by real-time PCR at48h,72h and96h post-injection. The WSSV copiesinshrimp with chlorpyrifos-WSSV treatment were about4times more than that withethanol-WSSV treatment at72h post-injection. The WSSV copies in shrimp withchlorpyrifos-WSSV treatment were significantly increased at96h, which was4.9times of that with chlorpyrifos-WSSV treatment at72h, and5.9times of that withethanol-WSSV treatment at96h. The acetylcholinesterase (AchE) activity in shrimpmuscle under chlorpyrifos stress was20%lower than that in control group. This showedthat the proliferation rate of WSSV in shrimp increased rapidly under chlorpyrifos stress,which accelerated the mortality rate of the shrimp.To investigate the toxicological mechanism of chlorpyrifos on the cultured shrimpLitopenaeus vannamei, the effects of sublethal dose of chlorpyrifos on immune-relatedgene expression have been invetigated according to the point of view of immunology.Firstly based on the acute toxicity tests, a chlorpyrifos concentration of0.38μg/L wasselected as the test concentration. AChE activities of gill and muscle in shrimp wereanalyzed. Temporalexpression levels of immune-related gene (PO, SOD, CAT, LyZ andPenaeidin-3a) were determined by Quantitative RT-PCR. The results demonstrated thatno mortalities were observed during the test period, and the shrimp exposed to thisconcentration of chlorpyrifos didn’t show the typical toxicity symptoms (like rapid anderratic swimming, shaking, loss of coordination and imbalance). Furthermore, the AChEactivities in gill and muscle of shrimp didn’t show the significant difference comparedto the control group after exposure to chlorpyrifos at24h,48h,72h,96h, respectively,except that AChE activity in gill at96h was inhibited approximately15%. PO mRNAexpression level was up-regulated significantly in the chlorpyrifos treatment group,which was1.38fold,1.54fold,1.95fold and1.40fold of PO expression levels in thecontrol group at0h,24h,48h,72h,96h, respectively. SOD and Penaeidin-3a shared thesimilar expression patterns. Their expression levels started to increase at24h andreached the peak at48h, which was2.5fold and2.14fold higher than that in the controlgroup, respectively, then decreased gradually to the normal level. LyZ mRNAtranscriptional levels initially rose up to the highest level from24h to72h, which was2.24fold higher than that in the control group. CAT mRNA expression levelsincreasedfrom24h to72h, and reached the highest value at72h, which was1.66foldhigher than that in the control group,then gradually decreased.In conclusion, chlorpyrifos was highly toxic for both juvenile and adult shrimpLitopenaeus vannamei. But it showed the stronger toxicity to juvenile shrimpunder thesame conditions. The mortalities of shrimp infected with WSSV significantly increasedunder chlorpyrifos stress, it suggested that the residues of chlorpyrifos could affect the immunity and it is necessary to supervise, the application of chlorpyrifos so that theconcentration of chlorpyrifos in aquatic environment was safe enough for culturedorganisms. In addition, some immune-relatedgenes were upregulated by sublethal doseof chlorpyrifos, which provided some helpful data for the toxicological mechanismresearch.