Identification Of Glutathione S-transferase Genes In Leptinotarsa Decemlineata And Their Responses To Three Pesticides

The Colorado potato beetle Leptinotarsa decemlineata(Say)is a notorious defoliator of potato.The beetles often cause extremely large potato yield losses.Historical reliance on insecticides has led to the rapid development of resistance in China populations,making it difficult to control.Synergism studies using diethylmeleate,an inhibitor of glugathione S-transferase(GST),showed that GST partially confers the resistance to phosalone and permethrin in several resistant strains.Moreover,our previous results reveal that diethylmeleate shows synergism to various insecticides in L.decemlineata.These data suggest that GST are involved in insecticide detoxification.However,the specific GST members remain unidentified.1.Identification of GSTIn the present study,the L.decemlineata genome and transcriptome were mined for putative GST-encoding transcripts.The resultant sequences were considered as L.decemlineata GST candidates(LdGST).The correctness of the putative LdGST sequences was substantiated by polymerase chain reaction(PCR).This was followed by 5'-and 3'-RACE to complete the sequence.The resulting sequence was submitted to GenBank(KU522306-KU522339).A maximum-likelihood(ML)tree was constructed using the annotated GST from T.castaneum,B.mori,A.mellifera,A.gambiae and A.pisum,as well as those from L.decemlineata.A total of 30 GST genes were identified.These GST belonged to cytosolic(29 genes)and microsomal(1 gene)classes.Of the 29 cytosolic LdGST,3,10,5,4,4,and 1 were classified as delta,epsilon,omega,sigma,theta,and zeta subclasses,along with 2 unclassified genes.2.Genomic organization of LdGSTThirty GST genes were mapped to 17 L.decemlineata scaffolds.Among 10 epsilon GST genes,8 LdGSTe genes(LdGSTe1 to LdGSTe8)were discovered on scaffold 12 and 2 LdGSTes(LdGST9 and LdGSTe10)were on scaffold 1223.The location of L.decemlineata epsilon GST genes on chromosome scaffolds is consistent with the hypothetical expansion history from the phylogenetic tree.Two omega and one sigma GST were located on scaffold 134.On scaffold 521,3 sigma members were discovered.The two unclassified GST members were located on scaffold 615.The other GST members were individually distributed on different scaffolds.The number of GST introns is thought to be associated with the ability to respond to xenobiotics and endogenous compounds.Therefore,the intron-exon structure of L.decemlineata GST were analyzed.A total of 83 introns were identified in the 30 LdGST.The average intron number was 2.8 for L.decemlineata GST genes.No intronless gene was found.In addition,we found that LdGSTe2,LdGST4 and LdGSTo3 possessed several splicing isoforms.3.The expression patterns under stress of three insecticides cyhalothrin,fipronil and endosulfan.In the L.decemlineata survivors suffered from cyhalothrin exposure,the expression levels of LdGSTe2a,LdGSTe2b,LdGSTe10,LdGSTu2,LdGSTo5,and LdGSTt1 were respectively upregulated by 18.0-,9.8-,3.4-,11.8-,4.9-and 21.4-fold.Similarly,fipronil treatment significantly increased the mRNA levels of LdGSTe2a,LdGSTe2b,LdGSTo5 and LdGSTt1 by 3.6-,28.3-,4.8-and 6.6-fold.Endosulfan application significantly augmented the transcript levels of LdGSTdl,LdGSTe2a,LdGSTe2b,LdGSTu2,LdGSTo5 and LdGSTtl by 4.9-,5.9-,13.8-,9.4-,12.0-and 4.1-fold.After cyhalothrin exposure,the mRNA levels of LdGSTe4 and LdGSTe6 were significantly decreased.Similarly,fipronil application significantly reduced the transcript levels of LdGSTe4,LdGSTe6 and LdGSTe10.Endosulfan treatment significantly lowered the expression levels of LdGSTe4,LdGSTe6 and LdGSTzl.