Reproductive And Detoxifying Enzyme Responses And Their Molecular Mechanisms Of Panonychus Citri (Acari:Tetranychidae) To Sulfoxaflor Stress

The citrus red mite,Panonychus citri（Mc Gregor）（Acari:Tetranychidae）is the most serious and persistent pest of citrus and navel oranges worldwide,mainly sucking the juice of leaves,green shoots and fruits with adult,larva and nymph mites,which seriously affects the yield and quality of citrus and navel oranges and other economic crops.The mite is a typical r strategy organism,with tiny individuals,high fecundity,short life cycle and easy to break out into disaster.Currently,chemical control is still the main strategy for citrus mite control.However,as the long-term continual abuse of chemicals,the citrus red mite has led to varying degrees of resistance to various insecticides.Sulfoxaflor as a neonicotinoid insecticide,has shown good control effect for many important cash crop pests with stinging-sucking mouthparts.However,there are no reports on the reproductive and detoxification enzyme responses of P.citri to sulfoxaflor stress and their molecular mechanisms.Therefore,in this study,we investigated the development and reproduction of P.citri at different concentrations of sulfoxaflor stress,and further explored the molecular mechanisms behind differences in fertility of P.citri.We analyzed the detoxifying enzyme response and molecular mechanism after insecticide stress on P.citri.The main results are as follows:1.Effects of sublethal concentration of sulfoxaflor on the growth,development and reproduction of P.citriIn this study,the toxicity of sulfoxaflor on the third instar adult female mite was determined by the leaf dish dipping method,and the results showed that the LC₅₀ value was 0.032 mg·L^-1,while the LC₃₀ value was 0.016 mg·L^-1.On this basis,we explored the growth,development and reproduction using the age-stage two-sex life table approach after sublethal concentrations of sulfoxaflor stress(LC₃₀ and LC₅₀).The experimental results showed that:From the parameters of growth and development,the incubation period,Larval period,nymph period,immature stage,adult life span and total life span of adult female and male mites were significantly lower in the LC₃₀treated group compared to the control group（P<0.05）.From the parameters of fecundity,compared with the control group,the LC₃₀ treated group showed a significant increase in egg production per female,V_xjvalues,m_x values and l_xm_x values at all ages（P<0.05）,while the LC₅₀ treated group showed a significant decrease in total pre-oviposition period,oviposition period,egg production per female,V_xj values,f_x5values and l_xm_x values at all ages（P<0.05）.From the parameters of population,compared with the control group,the net reproductive（R₀）and the intrinsic rate of increase（r_m）were significantly increased（P<0.05）,the mean generation period（T）and the population doubling time（t）were significantly decreased（P<0.05）in the LC₃₀treated group,while the net reproductive（R₀）and finite rate of increase（λ）were significantly decreased（P<0.05）and the population doubling time（t）was prolonged in the LC₅₀ treated group.The results of these experiments showed that sulfoxaflor at LC₃₀ concentration increased the fecundity of P.citri,which may cause the pest to become re-infested,while sulfoxaflor at LC₅₀ concentration shortened each developmental calendar and life span,reduced fecundity and significantly inhibited population growth.2.Effects of sublethal concentration of sulfoxaflor on the molecular mechanism of reproduction in the P.citriFrom the study in the previous chapter,it was found that sulfoxaflor at LC₃₀concentration significantly induced the fecundity of P.citri after stressing them,while sulfoxaflor at LC₅₀ concentration significantly inhibited their fecundity.Therefore,to further investigate the molecular mechanism of insecticide stress affecting the fertility of P.citri,the expression levels of vitellogenin（Vg）and vitellogenin receptor（Vg R）which are important genes related to reproduction and the content of Vg in P.citri were measured by RT-q PCR and ELISA respectively after 24h of sulfoxaflor stress with sublethal concentrations(LC₃₀ and LC₅₀).Experimental results showed that:compared with the control group,the content of Vg significantly increased 1.1 times（P<0.05）and the expression of Vg and Vg R were significantly increased 3.17 times and 1.5 times respectively after sulfoxaflor stress at LC₃₀ concentration（P<0.05）;while the content of Vg and the expression of Vg and Vg R were significantly decreased after sulfoxaflor stress at LC₅₀ concentration（P<0.05）.The results of these experiments showed that sulfoxaflor at LC₃₀concentration increased the fecundity of P.citri after stressing it,which may cause the pest outbreak,while sulfoxaflor at LC₅₀ concentration shortened each developmental calendar and lifespan,reduced fecundity,and significantly inhibited population growth after stressing P.citri.3.Detoxification enzyme response of P.citri to sublethal concentrations of sulfoxaflor stressChanges in the activity of detoxification enzymes（cytochrome P450,glutathione-S-transferase,carboxylesterase and acetylcholinesterase）and the expression of genes encoding them were determined by ELISA and RT-q PCR after sulfoxaflor stress on P.citri of adult female at sublethal concentrations(LC₃₀ and LC₅₀).The results of changes in target enzyme activities after sublethal concentrations of sulfoxaflor(LC₃₀ and LC₅₀)stress on P.citri was found:Compared with the control group,ACh E activity was significantly increased in the LC₃₀ treated group（P<0.05）;whereas ACh E activity was significantly decreased in the LC₅₀ treated group（P<0.05）.The results of changes in the main detoxifying enzymes activity were found:Compared with the control group,Car E,GSTs and CYP450 activities were significantly increased in the LC₃₀ treated group（P<0.05）;while CYP450 activities were significantly decreased in the LC₅₀treated group（P<0.05）and the changes in GSTs and Car E activities were not significant（P>0.05）.At the molecular level,the results of changes in the expression of genes encoding detoxification enzymes were found:Compared with the control group,the expression of Car E1 gene was significantly up-regulated（P<0.05）and the expression of GST gene was significantly down-regulated in the LC₃₀ treated group（P<0.05）.Compared with the control group,the expression of Car E7 and Car E9 genes were significantly down-regulated（P<0.05）and GST gene expression was significantly up-regulated in the LC₅₀ treated group（P<0.05）.The above results indicated that CYP450,GST,Car E and ACh E were all involved in the physiological process of detoxification metabolism of P.citri,and their enhanced activity and increased expression of encoded genes are important physiological,biochemical and molecular biological mechanisms of P.citri in response to sulfoxaflor stress;however,due to the different mechanisms of action of various enzymes,they play different roles in the detoxification of sulfoxaflor.4.Cloning of CYP2C15 and CYP391A1 genes and functional study of CYP392B6 gene in P.citriIn this study,the full-length of CYP2C15 and CYP391A1 genes from P.citri were cloned by RT-PCR combining with RACE technology.The c DNA of CYP2C15 gene is 1792bp,encoding 503 amino acids;The full-length c DNA of CYP391A1 gene is2083bp,encoding 617 amino acids.The amino acid sequence comparison showed that CYP2C15 was 61.46%similar to CYP2C15 of Tetranychus urticae and CYP391A1was 77.05%similar to CYP391A1 of T.cinnabarinus.The phylogenetic trees of CYP2C15 and CYP391A1 genes of P.citri and CYP450 genes of other species were constructed based on amino acid sequences using the neighbor-joining method,and the results showed that P.citri was most closely related to CYP2C15 of T.urticae,and P.citri was most closely related to CYP391A1 of T.cinnabarinus.To investigate the biological functions of the CYP392B6 gene of the P.citri,we analyzed the changes about the expression of CYP392B6 gene,the insecticide sensitivity and the content of cytochrome P450 in the P.citri of adult female after silencing the CYP392B6 gene by RNA interference technology.RT-q PCR results showed that the expression of CYP392B6 gene was significantly reduced 0.26 fold,0.57 fold and 0.50 fold when the concentration of target gene ds RNA was 1000 ng/u L,1500 ng/u L and 2000 ng/u L compared with the control group（P<0.05）.It can be seen that the best interference effect was achieved when the concentration of ds RNA was 1500 ng/u L.After effective silencing CYP392B6 gene of P.citri,the change of sensitivity was detected by stressing P.citri of adult female with sulfoxaflor at LC₅₀（0.032 mg/L）concentration and the results showed that the mortality of P.citri in ds RNA treated group was increased by17%compared with the control group,and the difference reached a significant level（P<0.05）.After effective silencing CYP392B6 gene of P.citri,the cytochrome P450content was significantly decreased in the ds RNA treated group compared with the control group（P<0.05）.In summary,this thesis investigated the effects of insecticide stress on the growth,development and reproduction of P.citri by using the age-stage two-sex life table approach,and analyzed the molecular mechanisms underlying the differences in their fecundity;Physiological and biochemical methods and RT-q PCR were respectively used to detect the activity of organismal detoxification enzymes and the expression of their coding genes after stressing P.citri with different sublethal concentrations of insecticides to investigate the relationship between insecticide stress and these detoxification enzymes.The molecular mechanism and response of P.citri to sublethal concentration of sulfoxaflor at the ecological,physiological,biochemical and genetic levels were revealed,providing a theoretical basis for the scientific control of P.citri and the rational use of sulfoxaflor.