| The small brown planthopper(SBPH),Laodelphax striatellus(Fallén),is an important pest widely distributed in rice producing areas in China,which has strong reproductive ability.Its nymphs and adults can pierce and suck rice juice through piercing-sucking mouthparts,laying eggs in rice stems and harming rice.Meanwhile,it can also spread a variety of plant virus diseases,such as rice stripe disease and rice black-streaked dwarf disease,which seriously affect the growth and yield of rice.Nowadays,the populations of SBPH in many areas have high drug resistance,and studies have proved that drug resistance and fecundity are largely related to lipid metabolism in insects.Lipid metabolism may play a key role in the large-scale outbreak of SBPH,and AKH signaling pathway is an important regulatory mechanism of lipid metabolism in insects,so it is of great significance to explore the role of AKH(adipokinetic hormone)signaling pathway in controlling SBPH.Wolbachia is a symbiotic bacterium widely existing in arthropods,which can regulate the reproduction,immune and nutrients of host insects.In the previous studies of our research team,it was found that there were significant differences in LsAKHR(adipokinetic hormone receptor)expression between Wolbachia-infected and Wolbachia-uninfected SBPH.In addition,some studies showed that Wolbachia could affect the resistance of host insects such as drug resistance and high temperature resistance.Therefore,this study aims to explore the effects of Wolbachia on lipid content,stress resistance of host SBPH from the perspective of AKH signaling pathway,so we can clarify the relationship between symbiotic bacteria Wolbachia and host insects,and provide a new theoretical basis for the control of SBPH.1.Effects of Wolbachia infection on survival rate and triglyceride content of SBPH after starvation and low temperature stressIn order to clarify the effect of Wolbachia on stress resistance of host SBPH,the resistance to hunger and low temperature of the Wolbachia-infected and Wolbachia-uninfected strains of SBPH were compared.The results showed that Wolbachia-infected can affect the resistance to hunger of SBPH.Compared with Wolbachia-uninfected SBPH,the starvation tolerance of infected adults was significantly improved and the survival time was longer;however,there was no significant difference between Wolbachia-infected and Wolbachia-uninfected SBPH at different-day ages.Wolbachiainfected can lesser affect the resistance to low temperature of SBPH.Under low temperature stress,compared with Wolbachia-uninfected females and 3th instar nymph,Wolbachia-infected individuals had significantly improved cold tolerance and longer survival time.However,there was no significant difference between other nymphs and male adults at different-day ages,nor between Wolbachiainfected and Wolbachia-uninfected SBPH.Then,the content of triglyceride in Wolbachia-infected and Wolbachia-uninfected SBPH was determined.Wolbachia-infected could increase the content of triglyceride of SBPH to some extent.The results showed that there was no significant difference in the content of triglyceride between the1 st and 4th instar nymph of the two strains,but the content of triglyceride in the 5th instar nymph,female and male adults of the infected strains was significantly higher than that of the non-infected strains.Moreover,when adults underwent low temperature and starvation stress,the triglyceride content of infected individuals was higher than that of uninfected individuals with time extension.Wolbachia-infected SBPH has higher triglyceride content,which may be related to its protective effect on host insects under stress.2.Cloning of AKH and its receptors c DNA gene sequences and analysis of their expression patterns in Laodelphax striatellus adultsBased on transcriptome data of SBPH,the c DNA sequences of AKH and its receptor(LsAKH and LsAKHR)were successfully cloned by PCR.Through sequence analysis,it was found that the c DNA sequence of LsAKH was 204 bp,encoding 67 amino acid,and had the conserved structural characteristics of neuropeptide.The conserved structure was “signal peptide + mature peptide + related peptide”,and the mature peptide sequence of LsAKH is “QVNFSPNWamide”.Phylogenetic analysis showed that LsAKH gene of SBPH was closely related to Nl AKH of Nilaparvata lugens(Stal),but far from AKH gene of Bombyx mori,Aedes aegypti and Culex pipiens.The c DNA sequence of LsAKHR is 1173 bp and encodes 361 amino acid.The protein domain analysis showed that LsAKHR had 7typical highly conserved transmembrane domains and belongs to typical G protein coupled receptor(GPCR).Phylogenetic analysis showed that the result similar with LsAKH gene,LsAKHR gene of SBPH was closely related to Nl AKHR gene of Nilaparvata lugens,but far from Dm Crz R(Corazonin receptor)of Drosophila melanogaster.The expression patterns of LsAKH and LsAKHR in adults of SBPH at different-day ages were analyzed by q PCR.The results showed that the expression of LsAKH was significantly high in 1-dayold adults of infected strains,and then low in female adults of infected strains with growth and development,but it gradually increased in the later stage of female adults.In summary,there was no significant and steady difference in the expression levels of LsAKH or LsAKHR in the two strains under normal feeding conditions.However,the differences of some individuals may be related that Wolbachia is involved in growth process.3.Expression pattern analysis and RNA interference analysis of LsAKH and LsAKHR in SBPH under low temperature and starvation stressThe expression patterns of LsAKH and LsAKHR in 3-day-old male and female adults of two SBPH strains under starvation and low temperature(4°C)stress were determined.The results showed that the expressions of LsAKH and LsAKHR showed a downward trend with the prolongation of starvation stress time,and the infected strains showed significantly lower expressions than the non-infected strains after 48 h of starvation.Under low temperature stress,the expression levels of LsAKH and LsAKHR also showed a downward trend with the extension of low temperature stress time,and the infected strain was significantly lower than the non-infected strain after 5 days of low temperature stress.At the same time,the down-regulation trend of LsAKH and LsAKHR expressions under starvation and low temperature stress consistent with the up-regulation trend of triglyceride content in Chapter 2,indicating that the triglyceride content in BPH may be regulated by AKH signaling pathway under stress.Using RNA interference technology,the dsRNA of LsAKHR gene was fed to female adults of SBPH,after feeding for 24 h and 48 h,compared with female adults of SBPH fed with ds GFP,the silencing efficiency reached 40% and 47% respectively.After interference,the triglyceride content in SBPH was detected,and it was found that the triglyceride content in infected and uninfected SBPH increased,and there was no significant difference.This phenomenon further indicated that Wolbachia might regulate the triglyceride content in host SBPH through AKH signaling pathway,thus improving the stress resistance of insects. |
