Acetylcholinesterase gene expression studies on sonata (fungicide) induced stress in channa punctatus and macrobrachium rosenbergii

Abstract

Acute toxicity tests (48hr, 72hr, 96hr LC50& Lethal concentration) of Sonata were conducted with two species (fish and prawn) viz. Channa punctatus and Macrobrachium rosenbergii. Acetylcholinesterase (AChE) is a key enzyme in the nervous system. It terminates nerve impulses by catalysing the hydrolysis of neurotransmitter acetylcholine. After serving as a neurotransmitter, acetylcholine is hydrolyzed by acetylcholinesterase (AChE). Inhibition of AChE is considered to be a specific biomarker for exposure to fungicides. In the present study, Acetylcholinesterase AChE genes were used in Channa punctatus and Macrobrachium rosenbergii brain were analyzed using real-time Polymerase chain reaction PCR to determine alterations in gene expression levels after Sonata fungicide treatment.  The AChE gene was isolated from fish and prawn brain by RT-PCR methods using degenerate primers.RNA isolated from two samples of Channa punctatus and Macrobrachium rosenbergii and they were subjected to agarose gel electrophoresis.. cDNA (Complementary DNA) will be synthesized from RNA and complementary DNA will be subjected to  RT-PCR. The amplified fragment of 800 nucleotides generated by PCR was cloned, and sequence analysis showed 80% nucleotide identity with AChE of the Electrophorus electricus. Specific primers of the fish and prawn AChE gene were then synthesized and used in the examination of AChE gene expression in brain tissue of fish and prawn exposed to sub lethal concentrations of sonata (36.05, 18.19 and 15.39ppm) and (18.40, 15.39 and 12.09ppm) for 48 hr, 72hr and 96 hr. RT-PCR was used to compare with the amplified GAPDH gene. Acetylcholinesterase gene expression got down regulated in Channa punctatus and up regulated in Macrobrachium rosenbergii after exposing of fungicides in comparison to the control group.