The Effects Of Pymetrozine And Other Insecticides On The Feeding And Reproduction Of Laodelphax Striatellus,and It’s Prelininary Mechanisms

The small brown planthopper,Laodelphax striatellus（Fallén）is an important pest,with wide distribution from eastern Asia to Southeast Asia,Europe and North Africa.Recently,the population density of the L.striatellus increased notably in China,especially in the middle and downstream Yangtze River and the northern rice regions.L.striatellus is an oligophagous pest,which had caused a serious damamge to many kinds of crops,such as rice,wheat and maize etc.Except for direct feeding damage,it can also transmit rice striped virus（RSV）,black streaked dwarf virus（BSDV）and maize rough dwarf virus（MRDV）,resulting in greater economic losses.With large-scale use of pesticides,it had been reported that L.striatellus had developed resistance to a lot of insecticides,such as imidacloprid,chlorpyrifos,deltamethrin,buprofezin and pyrethroid etc.However,chemical control was still a main method for controlling planthoppers in subtropical and temperate zones that was included China,Japan and South Korea.Therefore,it is necessary to find a relatively safe and effective insecticide.As a phloem-sap sucking pests,L.striatellus mainly secreted two kinds of saliva during feeding:gel saliva and watery saliva.Insect saliva has played a criual role in helping feeding,supressing plant defenses and regulating plant-insect interaction.However,the components of salivary proteins in L.striatellus and their functions in insect-plant interaction are still largely unknown.In the present paper,we studied the effects of five different insecticides on the development and reproduction of L.striatellus,detected the components of salivary proteins,and analysed the salivary glands of L.striatellus treated with pymetrozine by means of transcriptome and proteome.1.Effects of different insecticides on the development and reproduction of L.striatellus when the 3^rdinstar nymphs and the adults treated with these insecticides respectivelyFive insecticides（chlorpyrifos,imidacloprid,thiamethoxam,buprofezin and pymetrozine）were selected to study their effects on the development and reproduction of susceptible populations in Yunnan of L.striatellus.The results showed that no matter the 3^rdinstar nymphs or the newly emerged adults treated with insecticides,the developmental duration of F₁generation was prolonged,and longevity of female,oviposition duration,average eggs per female and valid eggs per female in F₀generation were reduced differently.Moreover,all of the five insecticides had a greater impact on adults in comparision with 3^rdinstar nymphs,with total eggs per female and valid eggs per female decreasing dramatically,especially the adult group treated with pymetrozine,the hatchability was much lower.2.Effects of pymetrozine at two different concentrations on the development and reproduction of L.striatellus when the adults treated with pymetrozineThe effects of pymetrozine at two different concentrations on the growth parameters of L.striatellus were studied for the first time.The results indicated that regardless of adults treated by 7 mg/L pymetrozine or 40 mg/L pymetrozine,the hatchability in F₀generation were both lower,and the depression effect was much more obviously in 40 mg/L pymetrozine treatment.According to the results of reciprocal cross,we concluded that pymetrozine had a greater effect on female adult than male adult,and pymetrozine sharply suppressed the fecundity of adults and the hatchability of female.3.Effects of pymetrozine on salivary glands,and components and function analyses of saliva secreted from L.striatellusThe effects of pymetrozine on the transcriptome and proteome of the salivary glands in L.striatellus were analysed in the paper.The transcriptomic and proteomic analysis of salivary glands were both summarized in three main categories:biological process,molecular function,and cellular component through Gene Ontology（GO）classification.Based on GO classification of differentially-expressed genes and proteins,it was figured out that the salivary gland of L.striatellus had much in common between transcriptome and proteome.Apparently,a gene product was likely to actively participate in one or more biological processes,act one or more molecular functions,and connect with one or several cellular components.Genes and proteins associated with“metabolic process”and“cellular process”accounted for the highest proportion in“biological process”,which were 28.16%and 22.85%of transcripts,and 32.26%and 27.42%of proteins,respectively.In addition,they both are involved in other biological process,such as biological regulation,localization,response to stimuli and signals.In“molecular function”,most of differentially-expressed genes and proteins in salivary gland involved in“catalytic activity”and“binding”,40.90%and 37.75%of transcripts,and 58.00%and 32.00%of proteins,respectively.In“cellular component”,differentially-expressed genes and proteins played a dominated role in“cell”（22.23%transcripts and 21.48%proteins）,“cell part”（22.28%transcripts and 21.21%proteins）,“membrane”（9.98%transcripts and 16.40%proteins）and“organelle”（15.83%transcripts and 11.79%proteins）.However,there were some differences in KEGG analysis between differentially expressed genes and proteins.KEGG analysis showed that differentially expressed genes in salivary glands mainly involved in carbon metabolism,amino acid biosynthesis,glycine,serine and threonine,and vitamin B6metabolic pathways.The majority of them performed in carbon metabolism（51.63%）,followed by amino acid biosynthesis pathway（32.11%）.Nevertheless,differentially expressed proteins chiefly distributed in lipid metabolism pathway,regulation of lipolysis in adipocyte and bile secretion pathway.It is the first time that we combined transcriptome and proteome of salivary glands in L.striatellus treated with pymetrozine.The transcriptomic data showed the function of salivary gland in m RNA level,while the proteomic data showed the function of salivary gland at protein level,which was more accurate and convincing.What’s more,the proteomic analysis maked some complement explanation and demonstration to the transcriptomic results of the salivary gland.According to the genome and transcriptome data of L.striatellus,a total of 211 proteins were identified from watery saliva in L.striatellus by shotgun LC-MS/MS analysis,73proteins contained signal peptide.Out of 211 proteins 92 proteins had 2-23 unique pep counts,but 119 proteins only contained one unique pep count.In the present study,watery saliva secreted from L.striatellus contained quantities of enzymes,such as oxidoreductases,hydrolases,lyases and ligases etc.Besides,binding proteins,transporter,salivary sheath protein,and other proteins without enzyme activity and unknown proteins were also found in the saliva.A total of 28 salivary protein genes of L.striatellus were selected for spatio-temporal expression analysis by quantitative real-time PCR（q RT-PCR）.Tissue expression analysis had revealed multiple expression patterns of salivary protein genes in L.striatellus.The transcripts of 13 genes,which encode six enzymes,five Ca²⁺-binding proteins and two mucins were detected highly expressed in salivary gland,showing that their corresponding proteins are mainly synthesized in salivary gland.So we hypothesize that those proteins play a role in the physiology of salivary glands or in secreted saliva for regulating host plant responses.Lysosomal aspartic protease（LAP）,beta-glucuronidase（GUS）,carboxylesterase,cathepsin B-like protease（CB-like）,trypsin-9 and trypsin-26were found in the saliva,but their transcripts were highly detected in the alimentary tract,showing their primary roles in digestion.The digestive enzymes that were secreted from salivary gland or gut played a role in extra-oral digestion of insects.To understand the functions of the salivary proteins,we aimed principally at the genes that are ubiquitously expressed in saliva among planthoppers by means of RNA interference（RNAi）approach.It was turned out that calmodulin（CaM）was essential for feeding.The interference of CaM resulted in their feeding time in nonpenetration and pathway phase increased dramatically,while feeding time in phloem and xylem decreased sharply,the honeydew area was also reduced a lot,as a result,the survival rate and the number of eggs were apparently reduced.However,the suppression of lipophorin precursor（LP）transcription levels caused no obvious difference in the feeding behavior and honeydew areas,while had a significantly negative impact on the survival rates,so we guessed that it was likely to affect survival rate of L.striatellus in other ways.