Response And Molecular Mechanism Of Panonychus Citri(Acari:Tetranychidae)to Spirobudiclofen Stress

Panonychus citri is a globally important agricultural pest mite,which is particularly harmful in citrus orchards,causing serious economic losses to the citrus industry.Currently,the mite is mainly controlled by the use of chemical insecticides,but the problem of resistance is becoming increasingly serious due to the frequent use of insecticides and the short developmental period and rapid reproduction of leaf mites.Insecticides not only kill pests directly,but sublethal doses of insecticides are also affect the population dynamics of insects by affecting their population parameters.Meanwhile,they may affect the composition and content of substance metabolism in the organism and changes in gene expression on certain metabolic pathways,etc.The present study was conducted to investigate the response of the P.citri to different sublethal concentrations of spirobudiclofen stress and its molecular mechanisms as a scientific problem:Firstly,the toxicity of spirobudiclofen to adult female was determined.On this basis,a life table was established to analyse the effects of different sublethal concentrations of spirobudicllofen stress on the growth,development and reproduction of their F1 offspring.The LC-MS technology and RNA-seq were used to analyse the lipid metabolomics changes and the differential gene expression levels of the corresponding metabolic pathways in adult female exposed to sublethal concentrations of spirobudiclofen.In addition,gene cloning,m RNA expression and RNAi technology were used to identify the key genes for lipid synthesis and transport and to verify their functions.The main results as follows:（1）Effects of different concentrations of spirobudiclofen on the growth,development and reproduction of P.citriAdults female were treated by leaf dipping method,and the female fertility life table was constructed to analyse the effects of different concentrations of spirobudiclofen(LC₃₀ and LC₅₀)on the growth,development and reproduction of their F1 generation.The results showed that compared with the control group,the immature stage（Egg,Nymph,Adults female）and lifespan of individual F1 generation in the acaricide treatment group were significantly shortened,and the shortening became more significant with increasing concentration.In addition,the oviposition period,pre-ovipositional period and total pre-ovipositional period were shorter in the spirobudiclofen-treated group compared with the control group,and the shortest spawning period was 6.62 d in the LC₅₀-treated group.Such as these population parameters:net population increment rate（R₀）,intrinsic growth rate（r）and weekly growth rate（λ）decreased with the increase of spirobudiclofen concentration,and the intrinsic growth rate decreased by 38%.These results showed that spirobudiclofen had a certain toxic effect on the adults female of P.citri,and shortened their life span and oviposition period to varying degrees,and reduced their oviposition.Therefore,it can inhibit the growth,development and reproduction of P.citri.（2）Metabolomics analysis of P.citri adults female after different concentrations of spirobudiclofenl stressedThe metabolomics of the proposed targeted lipids was analysed using LC-MS technology in adults female after exposure to different sublethal concentrations of spirobudiclofen.A total of 117 metabolites were found to be differentially expressed in the LC₃₀-treated group and 106 metabolites were found to be differentially expressed in the LC₅₀-treated group compared with the control group.Among these metabolites,81 metabolites（triglycerides,free fatty acids,etc.）were down-regulated in the two treatment groups with different sublethal concentrations of spirobudiclofen,while 12 metabolites（sphingolipids,acylcarnitine,etc.）were up-regulated.Amino acid metabolism,the metabolites that were significantly differentially altered in the spirobudiclofen treatment groups were enriched in the glycerophospholipid metabolism and amino acid metabolism pathways.（3）Transcriptomic analysis of P.citri adults female under different concentrations of spirobudiclofen stressedIn this study,Illumina RNA-seq sequencing technology was used to analyze the changes of metabolic pathways and gene expression of P.citri after the stress of spirobudiclofen.The results showed that:compared with the control group,there were 646 significantly differentially expressed genes in the LC₃₀ treatment group,564 significantly differentially expressed genes in the LC₅₀ treatment group.Among them,335 differentially expressed genes（such as Elongation of very long chain fatty acids protein,Ceramidase,Apolipoprotein D,etc.）were downregulated,while the other 10（such as ATP binding transporter G family member 20,etc.）were upregulated.In addition,compared with the control,the genes with common significant differences in LC₃₀ and LC₅₀ treatment groups were mainly enriched in sugar metabolism（amino sugar and nucleotide sugar metabolism）,amino acid metabolism（serine,threonine,etc.）and fat metabolism（fatty acid anabolism,glycerophospholipid metabolism,etc.）.Therefore,we thought that P.citri may respond to the stress of spirobudiclofen by regulating the key genes of lipid metabolism pathway.（4）Cloning and m RNA expression profile of lipid synthesis and transport genes of P.citriThe c DNA sequences of PcELO1,PcELO4,PcELO7,PcABCG20 and PcABCG23 were cloned for the first time,with full lengths of 1692 bp,1337 bp,1457 bp,3247 bp,and 2749 bp,respectively.The results of development specific m RNA expression showed that PcELO1,PcELO4,PcELO7 and PcABCG23expressed significantly higher in egg stage and adults female,while PcABCG20expressed relatively higher in egg and nymph stages.After adults female of P.citri were stressed with different sublethal concentrations of spirobudiclofefn,the results showed that the m RNA expression of PcELO7 and PcABCG23 decreased,and showed a certain concentration effect with the increase of concentration,indicating that P.citri may respond to spirobudiclofen stress by regulating the key genes（PcELO7 and PcABCG23）for epidermal lipid synthesis.（5）Using RNAi technology to explore the functions of PcELO7 and PcABCG23 genes involved in the development and reproduction of P.citriIn order to explore the biological function of PcELO7 in the growth and development of P.citri,this study conducted RNAi experiments on PcELO7 by feeding ds RNA to detect the silencing effect of P.citri adults female.The gene silencing effect was better after introducing 50 u L 2000 ng/u L ds PcELO7 into P.citri adults female for 48 h.Its lethal phenotype is mainly showed by body shrinkage and dryness.In terms of growth and development,compared with the control group,the survival rate,egg production,and hatching rate of adults female were significantly reduced.As the feeding time of ds RNA increased,the decrease became more significant.The m RNA expression levels of PcELO7 were significantly decreased.In the physiological and biochemical test results,it was found that the triglyceride content was reduced by 36%compared with the control.At the same time,oil red O staining showed that knockdown of PcELO7 significantly reduced the synthesis of fat in P.citri.The species and quantity of FFAs in adults female fed with ds RNA were studied by GC-MS.It was found that ds PcELO7 treatment significantly reduced the content of FFAs in adults female.Although there wee no significant difference in alkane species between the control and the treated groups,the number of alkanes in adults female was significantly lower in the treated group compared to the control.These results indicated that knockdown of PcELO7 had adverse effects on the growth and development and lipid synthesis of P.citri adults female.This gene may respond to the stress of spirobudiclofen by regulating the lipid synthesis process.In addition,this study further explored the biological function of PcABCG23 in the growth and development of P.citri.After ds PcABCG23（1500 ng/μL）was introduced into adults female by feeding method for 48 h,it was found that the gene silencing efficiency was relatively good.There were severe abnormal phenotypes in adults female after RNAi,including abnormal body shrinkage phenotype and small individuals who died due to inability to molt.In terms of growth and development,compared with the control group,the survival rate of adults female decreased significantly.With the increase of ds RNA introduction time,the decline was more significant,and the most significant decline occurred at 24 h and 48 h after ds RNA introduction.The hatching rate and fecundity of adults female were significantly decreased,and the decrease was more obvious with the increase of RNAi duration.The m RNA expression level of the target gene PcABCG23 also showed a significant decrease.At the physiological and biochemical level,we detected the triglyceride content of adults female introduced with ds PcABCG23 and found that the triglyceride content of the treatment group was reduced by 39%compared with the control.These results indicated that the knockdown of PcABCG23 had adverse effects on the growth and development and lipid transport process of adults female of P.citri.This gene may respond to the stress of spirobudiclofen by inhibiting lipid transport processes and reducing lipid accumulation in the cuticle.In conclusion,this study investigated the toxic effects of spirobudiclofen on P.citri adults female and molecular mechanism from the ecological,metabolomic and transcriptomic levels as well as the genetic level,which provided an important theoretical basis for the scientific control of P.citri and played a good guiding role in the research of RNAi molecular targets and further green pest control.