Identification And Pharmacological Characterisation Of Ionotropic ?-Aminobutyric Acid Receptor In Chilo Suppressalis(Walker)

The rice stem borer,Chilo suppressalis(Walker)(Lepidoptera:Crambidae)is one of the most damaging rice pests and occurs in all rice plant areas in China.To date,chemical insecticides control is still the major method to protect rice from damage by the rice stem borer.Unfortunately,C.suppressalis has developed resistance to many chemical insecticides,including chlorantraniliprole which just has been widely used to control rice pest in the last decade.Recently,rotation and mixture of different mode of action insecticides are the primary strategy employed to control C.suppressalis,and it is lack of single chemical insecticides to control the rice pest.Fluralaner,as a potent inhibitor of y-aminobutyric acid(GABA)receptor(GABAR),has been reported as ectoparasiticide.Moreover,fluralaner exhibits low toxicity or non-toxicity to mammals and fish.Therefore,it is expected to be used in the control of rice pests,but the specific site of fluralaner in GABAR is still unclear.Although the development of insecticides target on GABAR is rapidly progressing,the research on insect GABAR is relatively weak.To date,four GABAR-like subunits,including RDL(Resistance to dieldrin),LCCH3(Ligand-gated chloride channel homologue 3),GRD(GABA and glycine-like receptor of Drosophila)and 8916 subunits,are considered to exist in insects.However,only the RDL subunit is responsible for insecticidal resistance,the pharmacological characteristics of the other three subunits are still unclear.Therefore,in this study,we performed heterologous expression and functional studies to reveal the pharmacological characteristics of GABARs complex of different subunits from C.suppressalis.In addition,we used fixed-point mutation and electrophysiological assay to screen mutation types that are insensitive to fluralaner.It provides a evidence for the development and application of fluralaner,resistance monitoring,and development of novel insecticides target on GABAR.The results of this study are summarized as follows:1.Cloning and phylogenetic analysis of GABAR from C.suppressalis.GABARs play a variety of important physiological functions and is also one of the main targets of insecticides in insects.The GAB ARs composed of different subunits have different physiological functions and pharmacological properties.In order to clarify the type of GABAR subunits in C.suppressalis,we obtained two novel subunits of GABAR by searching the genome and transcriptome databases of C.suppressalis,namely CsRDLl(GenBank accession number:KX856969)and CsLCCH3(GenBank accession number:KX856968),respectively.Multiple protein sequences analysis result showed that the two subunits possessed common features of the dicysteine-loop ligandgated ion channel(cys-loop LGIC)superfamily.The same subunit type from different species of insects share didentity higher than 45%,while different subunit types from the same insect share didentity less than 25%,that indicates the different subunit types of GABARs with highly conserved functional domain but lower sequence similarity,whereas the functional domains and sequence similarity between the same type of subunits.Accordingto gene structure analysis,CsRDL1 and CsRDL2 are highly consistent in terms number and length of exon and intron phase,which implies that they are formed by the gene duplication event.Also,three pieces of alternative splicing were found in the CsRDL1 gene,forming eight different splice variants with different frequency,which greatly enriched the diversity of GABAR subunits in C.suppressalis.Phylogenetic analysis indicated that Lepidopteran insects usually possess multiple RDL subunits and lack the GRD subunits.Additionally,the insect GRD subunits and 8916 subunits are clustered with the vertebrate a and y subunits in the same clan,and the insect LCCH3 subunit are clustered with the vertebrate ? subunit inthesameclan.The important insecticide target RDL subunits in insects does not form a common evolutionary clan with any vertebrates specific subunits,indicating that the subunit composition of insect and vertebrate GABARs is species specificity,which provides a theory evidence for the development of highly selective pesticides.In summary,this study supplemented the type and quantity of GABAR subunits of C.suppressalis by comprehensive search of the genome and transcriptome data and found six different variants of CsRDLl by molecular cloning,which laid a theoretical foundation for further study of the pharmacological properties of the GABARs.2.Heterologous expression and functional analysis of the GABAR subunits from C.suppressalisPrevious studies have found that four subunits of RDL1,RDL2,LCCH3 and 8916 in Chilo suppressalis.To clarify the function of these subunits,we expressed these subunits and their mutants in Xenopus laevis oocytes,then,examined electrophysiological responses for agonist GABA and antagonist dieldrin,fipronil and fluralaner by two-electrode voltage-clamp(TEVC).The results showed that in vitro expressed CsRDLl and CsRDL2 and their mutants could form functional receptors independently.In contrast,CsLCCH3 and Cs8916 could not when singly injected.Both CsRDL1 and CsRDL2-S285A were sensitive to dieldrin with IC50 values of 86.4±21.5 and 353±108 nM,respectively.While CsCsRDLl-A282S and CsRDL2 showed resistance to dieldrin with IC50 values greater than 10,000nM.This indicated 2'serine cause insensitivity to dieldrin,but no significant effect on sensitivity of four subunits to fipronil and fluralaner.Co-injecting CsRDL1 and CsRDL2 with different ratios,all the receptors co-expressed showed more sensitivity to GABA and a significantly increased maximal current amplitudes than the alone injected with the same mass of CsRDLl or CsRDL2.The inhibition of dieldrin reduced with the increase of CsRDL2 ratio.Fipronil exhibited no significantly differences between the co-expressed receptors and singly expressed receptors,with IC50 values ranged from 5.05 to 6.65 nM,while fluralaner had a significant increased inhibitory with IC50 values ranged from 1.08 to 1.34 nM.Additionally,a functional receptor would assemble when CsLCCH3 and Cs8916 co-injected into the X.laevis oocytes,and gave a concentration-dependent current under GABA concentrations,with EC50 value of 22.51 ± 1.71 ?M and maximal current amplitudes of-166±44.4 nA.This co-expressed receptor was sensitive to dieldrin and fipronil,giving IC50 values of 5.91±2.08 and 6.31±3.8nM,respectively.However,it was insensitive to fluralaner with IC50 values greater than 10,000nM.These results provide a direct pharmacological evidence for demonstrating that the 8916 gene is a novel subunit of the GABAR.In summary,this study not only explored the relationship between the 2' amino acid residues and resistance,but also fully proved that different subunit types and the subunitratios can form GABARs with different pharmacological characterisation.These findings could not only instruct the development of novel insecticides,at the same time,it also laid a theoretical foundation for resistance management.3.Study on the toxicity and mechanism of fluralaner against C.suppressalisFluralaner is a novel class of insecticides acting on GABARs with a broad-spectrum activity and exhibits low toxicity or non-toxicity to mammals and fish.Therefore,it is expected to be used in the control of rice pests.The toxicity of fluralaner to C.suppressaliswere compared to fipronil performed by drop bioassay method.The results proved that fluralaner and fipronil had indeed similar toxicity to C.suppressalis.The previous study had demonstrated that fluralaner mainly acted on the RDL subunit of C.suppressalis.To clarify the key amino acid residue,the sequence of mammalian ?,? and ? subunits insensitive to fluralaner were aligned with those sensitive sequence of insect RDL subunits.The results showed there existed 35 different sites in the transmembrane region.Then,these specific sites that may contribute in insensitive to fluralaner were simulated and assessed by molecular docking.According to the results,12 amino acids at corresponding sites were mutated in vitro and injected into the X.laevis oocytes for electrophysiological assay.It was found that the mutation of CsRDL2-G319M is responsible for insensitivity to fluralaner by giving inhibition only 19.8±2.9%at the highest concentration of 10-5 M.And fipronil reduced the inhibition by about 2.2 times.However,the inhibitory of avermectin and broflanilideon CsRDL2-G319M reduced significantly by giving the inhibition rate only 45.4±4.4%and 24.5±1.8%under their highest concentration of 10-5M,respectively.Further,the G319M mutation in corresponding TM3 region third amino acid residue(3')was conducted into the RDL subunits of Laodelphax striatellus,Apis mellifera,Tetranychus urticae and CsRDL1,the shift from sensitivity into resistance to fluralaner was detected,with all the IC50S greater than 10,000 nM,while fluralaner has a potent inhibitor of non-mutant RDL subunits,with all the IC50s less than 10 nM.The above results indicated that the 3'G is a key amino acid residue in fluralaner acting on RDL GABARs.If a mutation occurs in the 3'G position of the insect RDL subunit,it may cause the pest generate resistance to fluralaner.