Molecular Regulation Mechanism Of ETH Signaling Systems In Development And Reproduction Of Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis?Hendel?,is one of the most destructive pests of agriculture throughout multiple areas of the world.Owing to its widespread polyphagous,rapid expansion and development of insecticide resistance,control of this pest has become more and more difficult.Neuropeptides constitute important regulatory signals for various physiological processes via activating their specific G-protein coupled receptors?GPCRs?.The multiple functions of insect neuropeptides make them ideal potential targets for the development of novel insect control agents based upon interference with their endogenous activities.This dissertation mainly studies the following aspects:Based on the larvae sclerite structure of fruit fly,the larvae of B.dorsalis were used to separate three instars accurately.Based on the transcriptome and genome assembly datasets,we reported the Ecdysis Triggering Hormone?ETH?precursors in 88 Diptera species.Moreover,the ETH of B.dorsalis was predicted and annotated via homology searches.The sequences Ecdysis Triggering Hormone receptors?BdETHR-A and BdETHR-B?and their genome structures of B.dorsalis were reported.To confirm the ETH signal transmission system,a calcium reporter assays were performed with their endogenous ligands in Chinese hamster ovary?CHO?cells.The temporal and spatial distributions of BdETH and their receptors?BdETHR-A and BdETHR-B?transcripts were investigated by quantitative real-time PCR?qPCR?.The localization of BdETH-expressing Inka cells in the tracheal tissue of larvae and female adult of B.dorsalis were investigated using immunohistochemistry and Fluorescence in situ hybridization?FISH?.The physiological funtions of B.dorsalis ETH signaling systems were analysed by RNA interference?RNAi?,HPLC and behaviour observations.Moreover,3D structures of BdETH,BdETHR-A and BdETHR-B was constructed using homology modeling,molecular dynamics simulation.Then BdETHR-A and BdETHR-B were used as target with mature peptides BdETH1 and BdETH2 for molecular blind docking.Finally,form the structure aspects,the binding sites were visualized for BdETH1-BdETHR-A,BdETH2-BdETHR-A,BdETH1-BdETHR-B,BdETH2-BdETHR-B by blind docking analysis.Our research lay a foundation for B.dorsalis control via designing newly agonists and antagonists.The main results in this thesis are as follow:1 Formulation of the criteria for the division of the larval instars of B.dorsalisThis study aimed to investigate the criteria for dividing the instars of B.dorsalis,which will be fundamental for understanding the molecular mechanism of larval growth and development for B.dorsalis.Five morphological variables,including the body length,the length and width of mouth hooks,and the length and width of the pharyngeal sclerite of the larvae,were measured.The Crosby growth rule was used in determining that B.dorsalis has 3 instars.The length of the pharyngeal sclerite is the best morphological variable for distinguishing the instars of B.dorsalis,whereas the length and the width of mouth hooks and the width of the pharyngeal sclerite can be used as additional characteristics.There was an overlap in the body length between the adjacent instars of B.dorsalis,and therefore body length cannot be used to separate the instars accurately.The objective of this study was to find a scientific,accurate,simple,and fast method that can be used as a classification criterion of instars of B.dorsalis,and we measured larval body length,the length and width of mouth hooks,and the length and width of the pharyngeal sclerite of B.dorsalis.2 The annotation,cloning,and identification of ETH signaling systemsBased on InsectBase,I5k genome and 1KITE database,we annotated ETH precursor sequences from 96 Diptera species.As indicated by the results,most neuropeptide precursors have conserved structures“signal peptide+mature peptides 1+spacer regions 2”.But,we find nine species,including Glossina 5 species,Mycetophila sp.,Megaselia abdita,Haematopota and Clusia lateralis,only has mature ETH1 and mature ETH2 is missing.The dibasic cleavage sites general were combinations of lysine residue?K?and arginine residue?R?in the two flanks of peptides.The most mature peptides have typical amidated C-terminnus motif KxxkxxPRx amide?X represents any amino acid?.The annotation of ETH precursor genes provides basic bioinformatics data for the functional study of the ETH signaling system in Diptera species.The full ORFs of BdETH precursors and their receptors?BdETHR-A and BdETHR-B?were amplified with nested PCR.Two B.dorsalis ETH peptides were predicted and two mature ETHs?BdETH1 and BdETH2?of B.dorsalis possess the general C-terminal motif sequence XXPRLXamide.Moreover,Both BdETHR-A and BdETHR-B are the typical GPCRs with seven transmembrane domains.An alignment between BdETHR and other ETHR sequences was performed,and the result showed that maximum similarity among these GPCRs essentially concerns the regions encompassing the transmembrane domains.It shows that ETH signaling syatem have simialr molecular mechanism regulating insect physiology.The phylogenetic tree was constructed with the splicing sites for phylogenetic analysis.The phylogenetic analysis indicated that the two receptor isoforms are separated into two groups,and that each is closely related to ETH-R-A and ETH-R-B from other Diptera,such as D.melanogaster and Aedes aegypti.To confirm that BdETHR-A and BdETHR-B are functional,a calcium reporter assay was performed with the two ETH peptides of B.dorsalis.The result shows that BdETH1 and BdETH2 both activated the BdETHR-A and BdETHR-B,expressed in CHO cells,and this in a concentration dependent manner.The BdETHR-A isoform was more sensitive to BdETH1 peptide?EC50=69±31 nM?compared to BdETH2?EC50=769±182 nM?,while BdETH1?EC50=39±16 nM?and BdETH2?EC50=30±11nM?activated the BdETHR-B.Based on median effective concentrations?EC50?,there was one difference that the BdETH2 peptide was?10-fold less active on the BdETHR-A than BdETH1,but for BdETHR-B,the two peptides could activate the receptor with similar EC50 values.3 Ecdysis triggering hormone signaling?ETH/ETHR-A?is required for the larva-larva ecdysis in B.dorsalisThe temporal and tissue-specific transcript profiles of BdETH,BdETH-R-A and BdETH-R-B were determined using RT-qPCR.BdETH was expressed in all developmental stages tested,but it was typical that for each instar that it showed a low expression at the early stage and a high expression at the late stage.This made its expression resemble a zigzag pattern.The highest levels of BdETH were recorded in the trachea.By immunohistochemistry?IHC?and fluorescent in situ hybridization,we localized the Inka cells that produce ETH in the tracheae of B.dorsalis at protein level and mRNA level.In order to determine the function of ETH/ETHR signaling in B.dorsalis,we downregulated the expression of ETH precursor transcript by RNAi and this resulted in the typical buttoned-up phenotype and tracheal defects in the 1^st and 2^nd instar larvae.In all cases,the buttoned-up phenotype resulted in ecdysis failure and the tracheal defects compromised the normal respiratory functions.The ecdysis failure contributed to the lethal phenotypes as affected larvae died soon afterwards.In our RNAi bioassays,we followed BdETHR-A and BdETHR-B and found that 1^st instar larvae treated with BdETH-dsRNA or BdETHR-A-dsRNA have similar phenotypes,while the 1^st instar larvae treated with GFP-dsRNA or BdETHR-B-dsRNA had no tracheal defects and ecdysis failure phenotypes.Taken all together,our data suggest that the BdETH signaling is essential for larval ecdysis in B.dorsalis,and that BdETH appears to activate a myriad of downstream signaling pathways via activation of BdETHR-A expression in the CNS.Overall,BdETHR-A expressing neurons are required for successful ecdysis throughout development of B.dorsalis.This study clarifies the functions of the ETH signaling pathway and suggests that the ETH/ETHR-A can be an effective target for the control of pest insects such as B.dorsalis.4 ETH signaling system mediates the fertility in the post-maturation female Bactrocera dorsalisBased on the qPCR,immunohistochemistry,fluorescent in situ hybridization,Calcium mobilization,RNAi and HPLC menthods,we measured relative levels of ETH,ETHR-A and ETHR-B expression in female adults.In general,the expression of ETH,ETHR-A and ETHR-B is in phase and resemble a zigzag pattern.Within 10 d after eclosion,the expression levels of ETH,ETHR-A and ETHR-B gradually decreased.Then,the expression level rises,until the fifteenth day reaches the peak.At protein and mRNA level,we localized the Inka cells that produce ETH in the tracheae of female B.dorsalis.The Inka cells localized at each branch point of the transverse connectives position of the tracheae in female B.dorsalis.We applied the calcium imaging to locate the presence of BdETHR transcript.The results show that exposure of CA excised to ETH1 and ETH2 mixture peptides produce robust calcium mobilization.These data confirm that CA is the mainly source of BdETHR in female adult B.dorsalis.In order to determine the function of ETH/ETHR signaling in female B.dorsalis,we downregulated the expression of BdETH or BdETHR by RNAi and this resulted in significant decrease of BdJHAMT and BdVg2 expression levels.We also measured the JH III titers of per female adult by HPLC method,when the BdETH or BdETHR was knocked down resulted in a significant decrease for JH III titers and the significant drop in egg production than control.BdETH/BdETHR or Vg2 expression was rescued to normal following injection of 0.5?g 20E or 0.5?g Met.When BdETH or BdETHR was knocked down,it led to a significant drop in egg production to normal levels.In conclusion,we provide model that endocrine network,20E,ETH signaling system,JH and vitellogenin,is involved in reproductive functions of the post-maturation female flies.Our study,as a whole,has shed light on the endocrine network-regulated reproduction in the post-maturation female of B.dorsalis,with potential target genes application to control for the control of pest insects such as B.dorsalis.5 The molecular docking analysis of ETH receptor and ligands interactionsBased on the homology modeling,molecular dynamics simulation and molecular blind docking analysis,3D structure of BdETH,BdETHR-A and BdETHR-B were constructed.Two mature peptides?BdETH1 and BdETH2?as the ligands and the BdETHR-A and BdETHR-B as the targets to run the blind docking analysis.We analyze the binding sites of BdETH1-BdETHR-A,BdETH1-BdETHR-B,BdETH2-BdETHR-A and BdETH2-BdETHR-B.The results show that the binding sites of BdETH1-BdETHR-A are R16-V67,S3-T73,N1 and E2-E70;the binding sites of BdETH2-BdETHR-AareS1-C15andL6-T45;thebindingsitesof BdETH1-BdETHR-B are L8-M41,F6 and F7-R42;the binding sites of BdETH2-BdETHR-B are K7 and T11-T22,I15-N51.The research can provide the basic information for designing agonists and antagonists targeting ETH receptors.In conclusion,the results provide abundant information for ETH signaling system and help better understanding the exact mode of action of ETH signaling system for influencing molting and reproductive behaviours,and lay a foundation for B.dorsalis control via development of agonists and antagonists.