Role Of IMD Pathway In Regulating Arsenophonus Population In Nilaparvata Lugens

Symbiotic bacteria are an important driver of biological evolution and widespread in nature.Symbiotic bacteria have an important influence on the nutrition,reproduction and immunity of their host insects.The role of IMD pathway,an important pathway for insects to defend themselves against Gram-negative bacterial infestation in regulating symbiont titer,is still laragely unknown.So in this experiment,the Nilaparvata lugens(N.lugens)and its internal symbiotic bacterium Candidatus Arsenophonus nilaparvatae(Arsenophonus)was used to investigate the effect of the IMD pathway on the titer of Arsenophonus using in protein expression,immunofluorescence and RNA interference techniques,and the following main results were obtained.1.Relish of N.lugens and Arsenophonus outer membrane protein A(Omp A)gene sequences were obtained in the N.lugens and Arsenophonus genomes respectively by homologous sequence alignment,and their complete ORF sequences were amplified using in PCR.Phylogenetic evolutionary tree analysis revealed that the Relish of N.lugens and Arsenophonus Omp A were related to Relish and Arsenophonus Omp A in other Hemiptera.The Relish of N.lugens has the conserved RHD and IPT structural domains shared by the Rel family at the N-terminal amino acid and several ANK structural domains at the C-terminus.Using Signal P and TMHMM online software,it was determined that Arsenophonus Omp A has a signal peptide and a transmembrane region structure.The Relish of N.lugens doesn’t have a signal peptide or transmembrane region.Arsenophonus Omp A was successfully expressed in E.coli by a prokaryotic expression system and a polyclonal antibody of good potency was prepared.2.Arsenophonus Omp A was localized in the fat body,intestine and ovaries of the N.lugens.In the intestine,Arsenophonus is distributed within the epithelial cells as well as between cells.In fat bodies,Arsenophonus may be present as aggregated balls or encapsulated by vesicles.In the early stages of N.lugens ovarian development,Arsenophonus is transferred from the fat body to the anterior part of the ovary and later enters the oocyte.At 0h,24 h,36h and 48 h of emergence,Arsenophonus Omp A is localised in the gonads at the tip of the ovarian tubules.At 72 h after emergence,Arsenophonus Omp A is labelled in the inverted oocyte of the ovarian tubule.3.Inhibition of PGRP-LC expression by gene silencing in Arsenophonus N.lugens leads to down-regulation of the expression of the transcription factor Relish and the antimicrobial peptide genes defensins and lugensins in the IMD pathway.Inhibition of the expression of the transcription factor Relish also suppressed the expression of PGRP-LC and the antimicrobial peptide genes defensins and lugensins.Since the N.lugens PGRP-LC protein has a transmembrane region structure,PGRP-LC can be involved in the IMD pathway as a pattern recognition receptor.Inhibition of PGRP-LB expression in N.lugens leads to up-regulation of PGRP-LC,Relish and some antimicrobial peptide genes,and the PGRP-LB protein has an amidase active structure,so PGRP-LB can play a negative regulatory role on the IMD pathway.4.Inhibition of PGRP-LC,Relish,defensin A and lugensin B gene expression in the IMD pathway of N.lugens by gene silencing techniques resulted in a significant decrease in the titer of Arsenophonus in N.lugens fat body,while inhibition of PGRP-LB gene expression resulted in a significant increase in the titer of Arsenophonus in N.lugens fat body;inhibition of Relish and PGRP-LB expression resulted in a significant up-regulation of Arsenophonus titer in the gut of brown planthopper.The coordination of PGRP-LC and PGRP-LB of IMD pathway in the brown planthopper regulates Arsenophonus titer in different tissues.The results of this study will help to reveal the interactions between insects and symbiotic bacteria,and provide new ideas and avenues for the development of new pest inhibitors targeting insect immunity.