Molecular Mechanisms Of Fecundity Adaptation Of Sogatella Furcifera To Response Triazophos Stress

Sogatella furcifera,the white-backed planthopper,is one of the most prevalent pests on rice.Its nymphys and adults damages the rice by sucking plant juices and laying eggs in the leaf sheath;it also can spread southern rice black-streaked dwarf virus,lead to an indirect hazard.S.furcifera is a typical r-selected insect that prone to disaster with high environmental adaptability and reproductive rate.In present,chemical control is the mainly control method in S.furcifera field management,but the long-term and irrational use of insecticides can resulted in the pest resistance evolution.Concurrently,the insecticides applied in the field may reduce to a sublethal concentration on pest with differences in personal contact over time that resulted in a sublethal effect,except for its lethal effiction.Studies have shown that sublethal concentrations of insecticides can stimulate the reproduction of insects,resulting in a resurgence in abundance.Enhancement of reproduction therefore may be an adaptive mechanism of pest to insecticide-based stress,but its molecular mechanism is unclear at present.Understanding the molecular mechanisms of insect adaptation to insecticide stress are helpful to characterize insect resurgence under insecticide pressure,provide theoretical guidance for field insecticide use,and develop new pest control methods for sustainable pest control.In this thesis,the S.furcifera were taken as the research object in focusing on the reproduction-related genes,to analyse their function in reproduction of S.furcifera and explore their relationship with insecticide stress,for understanding the molecular mechanisms of reproductive adaptation in S.furcifera response to insecticide stress.And the main results are as follows:1 Transcrip tome analysis of Sogatella furcifera under insecticide stressWe used transcriptome analysis to compare the expression patterns of resistance and stress-response genes in S.furcifera subjected to imidacloprid,deltamethrin,and triazophos,to determine the molecular mechanisms of resistance to these insecticides.After the low-quality sequences were removed,a total of 6.66 Gb data were obtained.After assembly and eliminating redundancy,40,597 unigenes were retained.The total length,average length,N50,and GC content were 38,797,336 bp,955 bp,1,741 bp,and 37.84%,respectively.A comparative analysis of gene expression under imidacloprid,deltamethrin,and triazophos stress revealed 1,123,841,and 316 upregulated unigenes,respectively.These upregulated genes under imidacloprid stress included seven P450s(two CYP2 clade,three CYP3 clade,and two CYP4 clade),one GST,one ABC transporter(ABCF),and seven Hsps(one Hsp90 and six Hsp70s);one P450(CYP3 clade),two ABC transporters(one ABCF and one ABCD),and one Hsp(Hsp90)under deltamethrin stress;and one P450(CYP3 clade)and one ABC transporter(ABCF)under triazophos stress.Also,deltamethrin and triazophos stress induced the transcription levels of TOR,S6K,Vg,and VgR;imidacloprid inhibited the transcription levels of FAMeT,S6K,and HMGR.Eighty unigenes were significantly up-regulated under the stress of the three insecticide treatments,including laminin,larval cuticle protein,and fasciclin,which are associated with epidermal formation.To verify whether these commonly up-regulated genes can also be induced to express under the stress of other insecticides,imidacloprid,triazophos,deltamethrin,thiamethoxam,pymetrozine,chlorpyrifos,and abamectin were applied in the same way for the 5th instar nymph.The results show that these commonly up-regulated genes cannot be induced by all insecticides.2 Cloning and functional study of SfVg and SfVgR in S.furciferaVitellogenin(Vg),the precursor of yolk protein which providing nutrition for ovarian development and egg maturation,is mainly synthesized in the fat bodies of insects and enters the ovary through endocytosis of the vitellogenin receptor(VgR).A Vg(SfVg),Vg-like(SfVg-like),and VgR(SfVgR)gene were identified from S.furcifera based on previous transcriptome data and recently published S.furcifera genome data.Their sequence accuracy was verified by RT-PCR and RACE technology.The complete OFR lengths of the three genes contained 6108,6060,and 5796 bp,that encoding 2035,2019,and 1931 amino acids were obtained,respectively.Conserved domain analysis revealed that SfVg has three conserved domains,while SfVg-like lost the DUF1943 domain present in traditional insect Vgs.Phylogenetic analysis showed that SfVg-like did not cluster with conventional Vgs in insects,but clustered with similar Vgs that also lost the DUF1943 domain in other insects.Spatiotemporal expression analysis shows that SfVg and SfVgR are mainly expressed in females and show a trend of first increasing and then decreasing.SfVg-like has a high transcription rate in females,including higher transcription in eggs and 2nd instar nymphs.SfVg and SfVg-like were most expressed in fat bodies while SfVgR showed its high expression level in ovaries.Targeted silencing of SfVg and SfVgR by RNAi technology caused the female ovary developing stop and "banana-forming"mature egg cells absence.The female fecundity and hatchability was significantly lower than those of the control group;Similar phenomenon was not observed in the SfVg-like silencing group.Enzyme-linked immunoassay analysis showed that Vg synthesis in the whole body of females was significantly reduced after SfVg silencing,whereas the Vg content did not significantly affected by SfVg-like and SfVgR silencing.Silencing SfVgR caused Vg content in female ovaries to be significantly lower than that in the control group,indicating that silencing SfVgR prevented Vg from entering the ovary.3 Cloning and functional study of JH signal pathway-related genes in S.furciferaThe annotation information of the transcriptome and genome of S.furcifera was examined and its sequence was verified by RT-PCR and RACE technology.Ten JH signaling pathway related genes of S.furcifera were cloned.They were named SfMevPPD,SfHMGR,SfIPPI,SfFPPSl,SfFPPS2,SfFPPS-like,SfJHAMT,SfFAMeT,SfMet,and SfKr-h1,respectively,based on the sequence similarity and conserved domain analysis.Phylogenetic analysis showed that these genes were most closely related to the corresponding genes of Laodelphax striatellus and Nilaparvata lugens,and they clustered into a large branch of the Hemiptera with the corresponding genes of other Hemiptera insects.Spatiotemporal expression analysis shows that most of the genes have higher expression levels in the 5th instar nymph and adult stage;SfHMGR,SfFPPS2,SfFPPS-like,SfJHAMT,SfFAMeT,and SfKr-hl also have higher expression levels in the egg stage.Tissue expression analysis found that SfIPPI,SfFPPS1,and SfJHAMT had higher expression in the head and integument;SfFPPS2 and SfMet had higher expression in the ovary;the other genes were highly expressed in the fat bodies.RNAi technology was further used to study the functions of these genes on the reproduction of S.furcifera.After the dsRNA injection,the silencing efficiency of the target gene reached 66.35-88.56%.The results of bioassay analysis showed that in the treatment group(injected with dsHMGR,dsIPPI,dsFPPS1,dsFPPS2,dsFPPS-like,dsJHAMT,dsFAMeT,dsMet,and dsKr-h1,respectively),the number of eggs was significantly lower than that of the control group(injected with dsGFP),and the female ovarian development and SfVg transcription levels were significantly inhibited by target gene RNAi.This indicates that these genes play an important role in the reproduction of S.furcifera.It was also found that the reproductive inhibitory effect on S.furcifera was not as significant as that of other genes in the dsJHAMT and dsFAMeT treatment groups.Further combined interference experiments showed that the combination silencing of SfJHAMT and SfFAMeT had a significantly higher effect on female reproduction than the two genes silenced separately,consistent with the results of measuring the transcription level of SfVg;however,this effect can be recovered to some extent by injecting in vitro juvenile hormone analogs,showing that the silencing of SfJHAMT and SfFAMeT affects the reproduction of S.furcifera by affecting JH biosynthesis.Also,after the target gene was silenced,we measured the effects on the transcription levels of 9 other JH signaling pathway-related genes.The results showed that these genes have a certain regulatory effect on each other,especially the upstream gene related to JH biosynthesis which had a significant regulatory effect on the downstream gene.4 Cloning and functional study of TOR signal pathway-related genes in S.furciferaThe keywords of this study were used to search the annotation information from the transcriptome and genome of S.furcifera,and the corresponding genes of N.lugens were used as a template for a homology search.Splicing the transcriptome data of S.furcifera was carried out using the software Geneious R9.Four TOR signaling pathway related genes of S.furcifera were obtained named as SfRheb,SfTOR,SfS6K,and Sf4EBPbased on their sequence similarity and conserved domain analysis.The sequences were verified by RT-PCR and RACE techniques.The complete OFR lengths of this four genes were 549,7488,1389,and 357 bp,encoding 182,2495,462,and 118 amino acids,respectively.Phylogenetic analysis showed that these genes were most related to the corresponding genes of the L.striatellus and N.lugens,and they cluster into a large branch of the Hemiptera separately with the corresponding genes of other Hemiptera insects.Spatiotemporal expression analysis results show that all genes have higher expression levels in the newly emerged female.SfRheb and Sf4EBP have the highest expression levels on the 4th day of 5th instar.Also,SfRheb,SfTOR,and SfS6K had a high level of expression in the head and integument,whereas Sf4EBP had a high level of expression in the integument and ovary.RNAi technology was further used to study the functions of these genes on the reproduction of S.furcifera.After dsRNA injection,the silencing efficiency of the target gene reached 73.91-86.09%.Bioassay analysis results showed that in the dsRheb.dsTOR,and dsS6K injection group,the number of eggs was 71.38%,51.78%,and 59.87%lower than that of the control group(injected with dsGFP)respectively;and those dsRNA injection treatment alsosignificantly inhibited female ovarian development and SfVg transcription levels.These results suggested that these genes play an important role in the reproduction of S.furcifera.The experiment using in vitro injection found that the effects of SfRheb and SfTOR silencing on S.furcifera spawning and ovarian development and SfVg expression can be restored to some extent by injection of JH analogs.After silencing,the SfRheb,SfTOR and SfS6K genes could significantly affect the transcription level of genes related to JH biosynthesis,further showing that SfRheb and SfTOR genes can regulate the reproduction of S.furcifera by affecting JH biosynthesis.5 The stimulate reproduction molecular mechanisms of triazophos stress on S.furciferaIn previous study of our research group,it was found that triazophos at a sublethal concentration can significantly induce the fecundity of S.furcifera,and thiamethoxam of sublethal concentration can significantly inhibit the fertility of S.furcifera.Therefore,based on previous studies,the expression level of reproduction-related genes in the female on the 3rd day of emergence was determined after 48 hours of stress on the 3rd instar nymph of S.furcifera with sublethal concentrations of triazophos and thiamethoxamine(LC10 and LC25).The results showed that triazophos LC25 treatment could significantly induce the transcription level of SfVg/SfVgR and increase the content of SfVg in the body of a female.The triazophos LC25 treatment also significantly induced the transcription level of genes related to the JH and TOR signaling pathways.Thiamethoxam stress resulted in the opposite results of the triazophos LC25 treatment,although the transcription level changes of some genes were inconsistent.Also,the LC10 treatment group had no significant effect on the transcription levels of these genes compared to CK,but there were significant differences compared to the LC25 treatment group,indicating that under different concentrations of insecticide stress,the reproductive regulation mechanisms of S.furcifera are different and there is a significant dose effect.Combined with the results of previous studies,it can be inferred that insecticides can regulate the JH signaling pathway by influencing the transcription levels of genes related to the TOR signaling pathway,thereby regulating the transcription level of SfVg,and ultimately regulating the reproduction of S.furcifera(stimulating or inhibiting reproduction).In summary,this thesis used triazophos stress to stimulate the reproduction of insects as an entry point and then screened genes related to reproduction with transcriptome sequencing technology.After S.furcifera was stressed with insecticides,the expression of reproductive-related genes was measured by RT-qPCR technology,and the relationship between insecticide stress and the expression of these genes was explored.The molecular mechanism of insecticides to stimulate the reproduction of S.furcifera was further clarified.The results of this thesis not only expand research on the molecular mechanisms of reproductive adaptation of insects in response to insecticide stress but also screen some key genes that regulate the reproduction of S.furcifera.Such results provide a framework to isolate targets for future insect pest control strategies.