Toxicity To Nilaparvata Lugens And Transport Mechanism Of Na~+/K~+-ATPase Inhibitors

Brown rice planthopper（BPH,Nilaparvata lugens St（?）l）is the important pest of rice in China,which seriously threatens the safe production of rice and causes huge economic losses to agriculture economy.Chemical control is still the main mean for the control of BPH.However,due to the long-term use of a large number of chemical pesticides,the resistance of BPH continues to inscrease,resulting in the control effect of chemical pesticides getting worse and worse.Therefore,the development of new target insecticides has become an urgent need for BPH control.Sodium potassium ATPase（Na⁺/K⁺-ATPase,NKA）is a membrane protein of eukaryotes.It maintains the transmembrane potential difference inside and outside the cell by transporting Na⁺and K⁺,which is very important for signal transduction and secondary transport in organisms.Previous studies in our laboratory found that after the interference of the NKA gene ATP1A1,planthoppers exhibited a high lethal phenotype,indicating that NKA is a potential target for the control of BPH.Therefore,the study on the toxicity of NKA inhibitors to BPH and their transport mechanism in BPH will be helpful for evaluating the potential of NKA as a new insecticide target.The main results are as follows:（1）Toxicity of NKA inhibitors to BPH.Two classical inhibitors of NKA,ouabain and digoxin,were selected to determine their toxicity to BPH by microinjection and artificial diet feeding,respectively.The results showed that ouabain and digoxin had very high toxicities to BPH.The toxicities of ouabain and digoxin to BPH were similar.Both of them exhibited significantly higher toxicities against BPH than chlorpyrifos.The toxicities of ouabian and digoxin determined by artificial diet feeding method were very different.The toxicity of digoxin of 10^-6M concentration is close to that of 10^-4M ouabain concentration.We speculate that the toxicity difference may be related to transporters.（2）Relationship between RNAi of Oatp genes and toxicity of NKA inhibitors.Organic anion transporting polypeptide（Oatp）belongs to the solute transport protein superfamily and is one key protein for cells to absorb foreign substances.There are six Oatp genes in BPH.The expression levels of Oatp genes in the head,midgut,hindgut and Malpighian tubes of BPH were detected by qRT-PCR.The results showed that the six Oatp genes were highly expressed in the intestines,among which the expression levels of Oatp58Da and Oatp58Db were the highest,and they were enriched in Malpighian tubes.The mortalities of BPH after RNAi with these six Oatp genes were low.Among them,only the mortalities of BPH after RNAi with Oatp1B2 and Oatp58Da genes were significantly different from that of the control group,with the adjusted mortalities of28.8%and 34.1%respectively.Four Oatp genes,Oatp1B2,Oatp74D,Oatp58Da and Oatp58Db,were selected for RNAi.The toxicities of non-lethal concentrations of NKA inhibitors to BPH after RNAi were measured by artificial diet feeding method.The results showed that RNAi of four genes significantly enhanced the feeding toxicity of ouabain to BPH;However,only the RNAi of Oatp58Da could significantly improve the feeding toxicity of digoxin to BPH.（3）Relationship between RNAi of Mdr genes and toxicity of NKA inhibitors.Multidrug transporter（Mdr）belongs to ABC transporter family,which is deeply involved in the detoxification mechanism of exogenous toxins.There are three Mdr genes in BPH.The expression levels of Oatp genes in the head,midgut,hindgut and Malpighian tubes of BPH were detected by qRT-PCR.The results show that the expression patterns of Mdr genes were consistent with Oatp genes.The three Mdr genes were highly expressed in the intestines,among which the expression levels of Mdr49 and Mdr50 were higher,and they were enriched in Malpighian tubes.After RNAi of three Mdr genes,there was no obvious lethal phenotype in BPH.The toxicities of non-lethal concentrations of NKA inhibitors to BPH after RNAi was measured by artificial diet feeding method.The results showed that the RNAi of Mdr49,Mdr50 and Mdr65 genes significantly enhanced the feeding toxicity of ouabain and digoxin to BPH.In conclusion,NKA inhibitors exhibited very high insecticidal activities against BPH,indicating that NKA has the potential as a target for the control of BPH;It was found that different NKA inhibitors had very significant differences of feeding toxicity to BPH,and it was speculated that different NKA inhibitors had different transport pathways in BPH.The RNAi of Oatp and Mdr genes had little effects on the survival rate of BPH,but they can significantly enhance the feeding toxicity of NKA inhibitors to BPH,which provides a new idea for the development of control strategy of BPH based on multi-target approaches.