Molecular responses and the toxicity of chlorpyrifos in giant tiger shrimp

Abstract

To examine the effects of chlorpyrifos to P. monodon, acute toxicity, inhibition of acetylcholinesterase activity, genotoxitity, and molecular responses of the exposed shrimps to chlorpyrifos were studied. Using static system, the LC50 values after 24 to 96 h of shrimp exposed to the lethal concentration of 68.1 nd 681 ug/l of chlorpyrifos were 1.7 and 3.3 times lower than that of control shrimp after 30 min exposure ((N=5, P<0.05). The enzyme activity in shrimp exposed to the sub-lethal concentration at 0.681 ug/l of chlorpyrifos was 1.9 times lower than that of control shrimp after 72 h exposure (N=5, P<0.05). The sensitive reduction of AChE activity at the sub-lethal concentration was 30 times lower than 96 h LC50 value, indicating the potential use as biomarker of chlorpyrifos exposure. The in vitro testing for genotoxicity using single-cell gel electrophoresis revealed significant increase of the DNA tail length and tail moments detected from the haemocytes exposed to 0.034 and 0.170 ug/l of chlorpyrifos in comparison with that of control group within 1 h (N=3, P<0.05) and the evidences were still detected after 6 h exposure to 0.170 ug/l of chlorpyrifos. Screening for candidate genes of the shrimp induced by chlorpyrifos exposure using mRNA DDRT-PCR revealed forty-four differential displayed transcripts. BLAST result identified 22 transcripts (16 up-regulated and 6 down-regulated) as known genes and 22 transcripts as unknown genes and genes similar to hypothetical proteins found in other species (8 up-regulated and 14 down-regulated). Expression analysis of well-characterized genes including cytochrome P450, beta glucuronidase, heat shock protein 70 heat shock protein 90, vitellogenin, and glutathione-s-transferase as well as genes obtained from mRNA DDRT-PCR, including OPA07G350-27-1 (LDL receptor member LR3), UBC101C-1,000-D-3 (esterase), UBC119A-650-F-5 (CYP330A1), OPA18G-600-4-1 (ubiquitin-like-7), OPA01G-425-1 (leucine zipper protein 5) and OPA02G-450-2 (sequence of unknown gene) were carried out using semi-quantitative RT-PCR. The results showed no significant difference in expression level among group of shrimp exposed to 0-27.24 ug/l chlorpyrifos within 96 h. Using RACE-PCR, full length cDNA of carboxylesterase (1,746 bp ORF encoding 582 amino acids), cytochrome P450 (1,530 bp ORF encoding 510 amino acids), and glutathione-s-transferase (654 bp ORF encoding 218 amino acids) were firstly reported. The results from this study provided basic information for toxicity of chlorpyrifos to P. monodon and revealed the potential use of acetylcholinesterase activities as biomarker for detecting the pesticide exposure.