Identification And Characterization Of Aminobutyric Acid Receptor Genes In Leptinotarsa Decemlineata

The Colorado potato beetle Leptinotarsa decemlineata(Say)is an invasive insect pest.It invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan.It is a notorious defoliator of potato throughout most of the northern Xinjiang in current,and often causes extremely large yield losses of potato.The control of this pest has mainly relied on insecticides.The y-aminobutyric acid(GABA)receptors are a type of ligand-gated ion channel receptors.They were the main inhibitory neurotransmitter GABA receptors in central nervous system of vertebrates and invertebrates.Based on L.decemlineata transcriptome and genome database,we abtained six GABA receptor cDNAs.We analyzed their expression responses to fipronil exposure,and explored their physiological roles by RNA interference(RNAi).The main results were shown as fellows.1.Molecular cloning of six GABA receptor genes in L.decemlineataSix GABA receptor gene fragments were mined from transcriptome and genome databases of L.decemlineata.The six receptor genes were classified into ionotropic receptor GABA-A and metabotropic receptor GABA-B.GABA-A group included Ld8916,LdGRD.LdGluCl and LdLCCH3.LdGABA-B1 and LdGABA-B2 belong to GABA-B.By RT-PCR,we confirmed the correctness of the six gene fragmens.Using the RACE technology,the full-length cDNAs of six genes were obtained.According to the results of multiple alignment and evolution analysis,we found these GABA receptor genes were highly conservative and they had the characteristic structures and domains of GABA receptors.It is suggested that these cDNAs may encode functional GABA receptors in L.decemlineata.2.The temporal and spatial expression profiles of six GABA receptor genes,and response to fipronil in L.decemlineataWe analyzed the expression of six GABA receptor genes in Malpighian tubules,brain,midgut,thoracic muscle,ventral ganglion,epidermis and fat body from day 1 fourth-instar larvae,the results showed that the mRNA levels of the six receptor genes were highly expressed in the brain and ventral ganglion.This expression profiles are consistent with the tissue-biased transcription of GABA receptor genes in other insect species.The fourth-instar larvae and adults were respectively exposed to sublethal doses of fipronil.For the fourth-instar larvae,they were treated at the 0 and 60 hour after molting.Fipronil exposure initiating at 0 h after ecdysis significantly downregulated the expression of Ld8916,LdGRD,LdLCCH3.LdGABA-B1 and LdGABA-B2.In contrast,fipronil treatment initiating at 60 h after ecdysis significantly upregulated the expression of Ld8916,LdGRD,LdGluCl,LdLCCH3,LdGABA-B1 and LdGABA-B2.The same exposure protocol was used to analysis newly-emerged male and female adults.The results indicated that after fipronil application,the expression levels of the six GABA receptor genes in adults significantly rose.It seems that all the six GABA receptor genes respond to fipronil in a developmental stage dependent way.3.RNA interference-mediated knockdown of the six GABA receptor genes in L.decemlineataTo investigate the physiological roles of the six GABA receptors in L.decemlineata,we dietarily introduced dsRNAs respectively derived from the six GABA receptor genes into the newly-molted fourth-instar larvae.After 3 days of ingestion,treated samples were used to extract total RNA.qRT-PCR was used to test the target genes transcription.The results showed that ingestion of dsRNAs could knock down their target genes.Moreover,feeding of dsLd8916,dsLdGluCl and dsLdLCCH3 for three days significantly dropped larval survival rate.In contrast,feeding each of dsRNAs from other three GABA receptor genes caused little larval mortalities.Furthermore,ingestion of dsLd8916 and dsLdGluCl resulted in approximately 60%of the larvae failure to pupate.The larvae finally died.Therefore,GABA receptors play important physiological roles in the survival and metamorphsis in L.decemlineata.