The Physiological Functions Of Tachykinin And Natalisin Signaling Systems And Assessment Of The Potential As Insecticidal Target In Bactrocera Dorsalis

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.In this study,based on the B.dorsalis transcriptome of central nervous system(CNS)and genome assembly datasets,the neuropeptide genes of B.dorsalis were predicted and annotated via homology searches.The sequences and structures of B.dorsalis tachykinin related-peptide(BdTRP)and natalisin(BdNTL)precursors and their receptors(BdTRPR and BdNTLR)were reported.The temporal and spatial distributions of BdNTL and BdTRP and their receptors transcripts were investigated by quantitative real-time PCR(qPCR).The localization of BdTRP-expressing and BdNTL-expressing neurones in the brains of B.dorsalis were investigated using immunohistochemistry and Fluorescence in situ hybridization(FISH).The physiological funtions of B.dorsalis TRP and NTL signaling systems were analysed by RNA interference(RNAi),antennal electrophysiological response and behaviour observations.Moreover,to confirm the B.dorsalis BdTRPR and BdNTLR are functional,a calcium reporter assays were performed with their endogenous ligands in Chinese hamster ovary(CHO)cells.Subsequently,the agonists and antagonists screening system for the BdTRPR and BdNTLR were established,and then,the pharmacological analyses were performed with peptidomimetics,which were confirm that they have agonistic and antagonistic activities.The main results in this thesis are as follow: 1 In silico identification and annotation of the neuropeptides for B.dorsalisBased on the B.dorsalis transcriptome of central nervous system(CNS)and genome assembly datasets,39 neuropeptide genes of B.dorsalis were identified and annotated via homology searches.As indicated by the results,most neuropeptide precursors have conserved structures “signal peptide + mature peptides + spacer regions ”.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.Moreover,part of peptides have N-terminally pyroglutamate block,such as adipokinetic hormone,allatostatin C,Corazonin and diuretic hormone 44.Some peptides have a disulfide bridge between the cysteines,like allatostatin C,allatostatin CC,crustacean cardioactive peptide,CCHamide,CNMamide,insulin-like peptide and Neuroparsin.Sulfakinin peptide possess a tyrosine sulfation site.The precursors of CNMamide,ion transport peptide and Orcokinin have alternative splicing and neuropeptide F precursor has gene duplication.The full ORFs of BdTRP and BdNTL precursors and their receptors were amplified with nested PCR.BdTRP and BdNTL precursors have conserved structures compared with other neuropeptides.Five B.dorsalis TRP peptides were predicted and four putative TRPs(BdTRP1-4)of B.dorsalis possess the general C-terminal motif sequence Fx Gx Ramide.B.dorsalis NTL precursor has three mature peptides and all the BdNTLs possess the general C-terminal motif sequence FxxxRamide.Moreover,Both BdTRPR and BdNTLR are the typical GPCRs with seven transmembrane domains.An alignment between BdTRPR and other TRPR sequences was performed,and the result showed that maximum similarity among these GPCRs essentially concerns the regions encompassing the transmembrane domains.An alignment between Bd NTLR and other NTLR sequences is in accordance with result of TRPRs.The phylogenetic tree indicates that NTLRs and TRPRs are clearly divided into two different groups.2 The physiological function of TRP signaling system in B.dorsalisThe temporal and spatial distributions of B.dorsalis BdTRP and BdTRPR transcripts by qPCR.The results showed that BdTRP and BdTRPR were ubiquitously expressed at all the tested developmental stages.Both BdTRP and BdTRPR were highly expressed at adult stages.For the tissue-specific expression profiling,Bd TRP transcript was detected classically in the CNS and midgut.Besides the CNS,midgut and hindgut,it was of interest that BdTRPR expression was also detected in the antennae.In the brains of B.dorsalis,localization of BdTRP was identified with immunohistochemistry and FISH.By immunohistochemistry using a rabbit antibody against L.migratoria TRP peptide,the abundant TRP-producing cells were detected in the brain of B.dorsalis.In the protocerebrum area,one pair of dorsolateral interneuron(DLI)and posterior dorsolateral protocerebrum(PDLP)neurons were stained.In the tritocerebrum area,there are clusters of approximately 7 tritocerebral neurons in each hemisphere.Moreover,one pair of neurons were stained in the subesophageal ganglion and abundant neuropils were stained with anti-LomTRP1 in superior median protocerebrum.Noteworthily,there were about nine neurons with a stable TRPs staining situated in the local interneurons(LNs)and with cell bodies lateral to the antennal lobe(AL).These results reduce significantly from those obtained using FISH.One pair of DLIs and two pairs of dorsolateral protocerebrum(DLP)neurons.Similarly,in accordance with results of immunohistochemistry,the BdTRP-expressing were detected in LNs.Moreover,there were far fewer NTL immunoreactive neurones in the larval brain than in the adult brain.Four pairs of neurons were stained using immunohistochemistry and only one pair of neurons were detected by FISH.In order to confirm the role of TRP signaling system in regulating the odor sensitivity in B.dorsalis,we analyzed the EAG and olfactory behavioral changes by knocking down the TRP precursor gene as well as its receptor using dsRNA injection.The knockdown efficiencies were significant with 62.3-68.4% for BdTRP and BdTRPR mRNA levels at 48 h post-injection of dsRNA.The electrophysiological data demonstrated that the silencing of BdTRP or Bd TRPR reduced the antennal responses of B.dorsalis to ethyl acetate.In accordance with this,the odor choice assay in a Y-tube showed that oriental fruit flies treated with BdTRP-dsRNA or BdTRPR-dsRNA had lost this aversive behavior for ethyl acetate,they showed random choice for two terminals.In conclusion,these data provided clear evidence that the TRP signaling system is involved in the regulation of olfactory sensitivity in B.dorsalis and confirmed its potential as a target for novel insecticides.3 The physiological function of NTL signaling system in B.dorsalisB.dorsalis BdNTL and Bd NTLR transcripts were investigated by qPCR.The results showed that both BdNTL and BdNTLR were highly expressed at the pupal and adult stages.Among the tissues tested,BdNTL transcript expression was limited to the CNS.However,in addition to CNS,BdNTLR was also moderately expressed in the hindgut,antennae,ovary and testes.In the adult brains of B.dorsalis,NTL-expressing neurones were confirmed with both immunohistochemistry and FISH in anterior dorsolateral interneurones(ADLI),inferior contralateral interneurones(ICLI)and DLP neurones.As indicated by the results of immunohistochemistry,thirteen pairs of adult neurons were detected by the antibody,including two pairs of DLP and small pars intercerebralis(sPI)neurons,seven pairs of PDLP neurons,one pair of ADLIs and ICLIs.The expression of BdNTL mRNA was detected in ADLI,ICLI and DLP neurons using FISH.Moreover,there were far fewer NTL-expressing neurones in the larval brain than in the adult brain.Immunohistochemical data confirmed that 6 pairs of immunoreactive neurons were stained.Two pairs of neurons were detected by FISH in larval brains.To reveal the functions of the NTL signalling system in B.dorsalis,RNAi mediated by dsRNA-NTL or dsRNA-NTLR injection was employed.The knockdown efficiencies were significant with 51.0-81.6% for BdNTL and BdNTLR mRNA levels at 48 h post-injection of dsRNA.The results showed that downregulation of Bd NTL precursor transcript expression by RNAi caused reduced mating frequencies in B.dorsalis.However,BdNTL-RNAi did not modify egg production of successfully mated flies and have no effect of testes and ovary development in B.dorsalis adults.Meanwhile,BdNTLR-RNAi could cause reduced mating frequencies in B.dorsalis.In conclusion,these experiments revealed that NTL signalling system is involved in the regulation of mating ability in B.dorsalis and may be a potential target for B.dorsalis control.4 The screening of agonists and antagonists for BdTRPR and BdNTLRTo confirm that the Bd TRPR and BdNTLR are functional,the calcium reporter assay of BdTRPR and BdNTLR transiently expressed in Chinese hamster ovary cells were performed,respectively.The 50% effective concentrations(EC50)values of all endogenous ligands were measured for BdTRPR and BdNTLR.Among them,BdTRP5 and BdNTL2 were the most active one,respectively.Therefore,Bd TRP5 was set as model ligand for BdTRPR and BdNTL2 was set as model ligand for BdNTLR.Interestingly,The cross interactions were observed of TRP and NTL signalling systems in the in vitro assay.At micromolar levels,BdTRP5 could activate BdNTLR and BdNTL2 also could activate BdTRPR.Subsequently,the agonists and antagonists screening system for the BdTRPR and BdNTLR were established based on the functional assay of receptors and their model ligands.Assays on the two receptors with 13 peptidomimetics were performed in the agonists and antagonists screening system.The results showed there were no peptidomimetics that effectively activated or inhibited BdNTLR.Nevertheless,the peptidomimetics 1888[?1]wp-4 and 2120[?1]wp-1 activated BdTRPR in a concentration-dependent manner,with the EC50 values of 4.9 nM and 268.8 nM,respectively.Moreover,the peptidomimetic 1887[?1]wp-3 inhibited BdTRPR in a concentration-dependent manner,with 50% inhibitory concentration value at 0.57?M.In conclusion,the results provide abundant information for insect neuropeptides and help better understanding the exact mode of action of neuropeptide signaling systems for influencing behaviours,and lay a foundation for B.dorsalis control via development of agonists and antagonists.