Wolbachia-Mediated Stress Resistance In Laodelphax Striatellus

Wolbachia are extremely common facultative symbiont found in insect pests,and this bacterial symbiont have been well known to play an important role in the evolution of insects.Besides the main function as reproductive manipulators,Wolbachia has also been demonstrated to protect their host against RNA viruses which can cause diseases in Dipteran insect pests.Other than these,whether there are other Wolbachia-mediated roles on stress resistence deserves further investigation.The small brown planthopper(SBPH),Laodelphax striatellus(Fallen)is a notorious pest of rice crops,it also harbours Wolbachia.In this study,we firstly studied the community structure of endosymbionts in L.striatellus,then we focused on the impact of Wolbachia on their hosts' susceptibility to insecticides or insect pathogens,and on theirs tolerance to heat or frost stress,in order to clarify the regulation of Wolbachia on stress resistance in their hosts.All the results will not only be beneficial to under the evolutionary relation between host and symbionts,but also help us to explore new methods to control the insect pests.1.Community structure of endosymbionts in L.striatellus and its difference caused by chemical insecticidesTo clarify the community structure of endosymbionts in L.striatellus,seven known species of symbionts in planthoppers were detected.The symbiont infection status in L.striatellus from 7 regions including Nanjing(NJ),Fengxian(FX),Rugao(RG),Jiangdu(JD),Jiarhu(JH),Wujing(WJ),Jiangsu province and Chuxiong(CX),Yunnan province were compared,and the symbiont infection in buprofezin-resistant(BR),chiorpyrifos-resistant(CR)and susceptible(S)strains of L.striatellus were also compared.At the same time,the Wolbachia density in L.striatellus strains with different resistance level to buprofezin were also measured.The study found that there were difference in the symbionts species among different populations ofL.striatellus,Wolbachia,Serratia and Acinetobacter were present in all the 7 populations,Arthrobacter was only detected in JD,JH and WJ populations,Arsenophonus was detected in NJ,JD,FX,RG and CX populations,Cardinium hertigii and Chryseobaterium were not found in all the tested individuals.For the composition of symbionts,eight types of symbiont combination were found in L.striatellus,and the number of combination types in the buprofezin-resistant strain from CX was more than that in the buprofezin-susceptible strain with the same genetic background.The Serratia single infection and Wolbachia+Serratia multiple infection frequencies in the BR strain were significantly higher than that observed in the BS strain.The genetic difference among different Wolbachia strains were determined in L.striatellus.The result showed that,based on the analysis of wsp gene,Wolbachia CX,NJ,FX and RG strains all were wStri strain which belonged to B Con group,supergroup,and futher the genotyping analysis of MLST also indicated that there was no genetic diference amomg them.The higher density of Wolbachia,the higher level of resistance to buprofezin in L.striatellus.All the results indicated that the community structure of endosymbionts might be related with ecological adaptability or insecticide resistance of L.striatellus.2.The removal of Wolbachia by antibiotics in L.striatellusTo obtain the Wolbachia-free strains of L.striatellus,the removal efficiency of tetracycline on Wolbachia in L.striatellus was determined by quantity PCR.Wolbachia-infecete L.striatellus were fed on rice seedlings treated with 2 mg/m1 tetracycline solutions,then the Wolbachia density in L.striatellus were detected in each generation.The results showed that Wolbachia in females and males of L.striatellus could be removed effectively by tetracycline.The Wolbachia density of L.striatellus significantly declined with tetracycline treatment for one generation,there was a sharp decline in Wolbachia density with tetracycline treatment for two generations,and the Wolbachia density was at a very low level with tetracycline treatment for 2 generations.After 5 generations of tetracycline treatment,the Wolbachia density remained at a very low level without antibiotics treatment in the following generations,there was still no recovery of Wolbachia infection in 1sth generation.The above results showed that the Wolbachia in L.striatellus could be removed effectively with 2 mg/ml tetracycline for 2 generations,and after stopping tetracycline treatment,it would not be recovered 10 generations later.This provided us a useful method to establish Wolbachia-free strains of L.striatellus.3.Effect of Wolbachia infection on stress resistance in L.striatellusTo find the effect of Wolbachia on stress resistance in L.striatellus,the resistance to chemical insecticides,insecticidal bacteria,high and low temperature of the Wolbachia.infected and Wolbachia-cured strains of L.striatellus were compared.The results showed that the resistance to chemical insecticides of L.striatellus were affected by Wolbachia,the sensitivity to buprofezin all increased and chlorpyrifos sensitivity all decreased when the Wolbachia were removed from both the resistant strains and the susceptible strains of L.striatellus,however the sensitivity to thiamethoxam and pymetrozine unchanged.The sensitivity to buprofezin increased in RG strains and the sensitivity to chlorpyrifos decreased in NJ strains when the Wolbachia were removed,the sensitivity to thiamethoxam and pymetrozine unchanged in all strains.The Wolbachia in L.striatellus eould increase the resistance to Serratia marcescens in some strains.The Wolbachia had no obvious effect on the hatching rate of L.striatellus under high temperatures,it could increase the survival rate of the nymphs to some extent under 35?.Whereas Wolbachia had no significant effect on the tolerance of the nymphs under the extreme low temperature.Overall,Wolbachia infection in L.striatellus could improve the stress resistance of its host.4.Wolbachia-mediated resistance to chemical insecticides in L.striatellusTo explore the roles of Wolbachia in resistance to chemical insecticides in L.striatellus,the CX-S,RG,FX and NJ strains with and without Wolbachia were used to hybridize and backcross in order to replace the genetic material of Wolbachia-infected L.striatellus,and twelve new strains which were only replaced with the host genetic materials and retain the Wolbachia were obtained.The insecticide resistance levels of all the Wolbachia-infected and Wolbachia-free strains with the same genetie background of hosts were compared.The Wolbachia can be stable in the host with new genetic background.The Wolbachia CX strain could significantly increase the resistance to buprofezin in FX and NJ strains of L.striatellus,and the Wolbachia RG?FX and NJ strains all could significantly increase the resistance to buprofezin in RG strains of L.striatellus.The Wolbachia RG strain significantly increase the resistance to chlorpyrifos in both CX and NJ strains of L.striatellus,and the Wolbachia FX strain also significantly increase the resistance to chlorpyrifos in NJ strain of L.striatellus.Therefore the role of Wolbachia on the resistance to chemical insecticide resistance in L.striatellus was affected by both the endosymbiont and the genetic characteristics of the host.5.The effects of Wolbachia on enzyme activity and P450 gene expression in L.striatellusTo clarify the mechanisms of Wolbachia-regulated resistance in L.striatellus,the activity of protective enzymes and detoxification enzymes and the expression of P450-related genes were determined in L.striatellus.The results showed that,after removing of Wolbachia from L.striatellus,the activities of superoxide dismutase(SOD)increased in RG line and decreased in FX line;the activities of peroxidase(POD)increased in CX-R2 and decreased in FX line;the activities of carboxylesterase(CarE)decreased in CX-R1,CX-R2,CX-R3 and FX;the activities of Glutathione S-transferase(GST)increased in CX-S and decreased in CX-R2;the activities of Microsomal mixed function oxidases(MFO)increased in CX-R1,CX-R2 and CX-R3 and decreased in CX-R3.The expression of P450 related genes was significantly upregulated in some lines of L.striatellus.The expression of P450 genes were affected by both the interaction of L.striatellus and Wolbachia.The Wolbachia may improve the host's stress resistance by providing detoxification for L.striatellus.