The Physiological Functions Of Adipokinetic Hormone And Corazonin Signaling Systems In The Oriential Fruit Fly,Bactrocera Dorsalis(Hendel)(Diptera:Tephritidae)

Neuropeptides play central roles in regulating numerous vital physiological systems and behavioral events in diverse insects,which make them a prime practical potential target in the development of novel insect control agents.The oriental fruit fly,Bactrocera dorsalis?Hendel??Diptera:Tephritidae?is a quarantine polyphagous pest and one of the most destructive pest of fruit industries due to its wide distribution and rapid invasiveness.Previous studies showed that adipokinetic hormone?AKH?and corazonin?Crz?play important roles in insects.Therefore,investigating the functions of AKH and Crz signaling systerms in B.dorsalis is important.This study will contribute to a better understanding of the AKH and Crz signaling processing in this agriculturally important pest insect,and may shed light on their potential as the target of novel insect control agents.In the present study,we identified and characterized the full-length cDNAs of AKH and Crz and their cognate receptor in B.dorsalis?BdAKH and BdAKHR,BdCrz and BdCrzR?;With transformed Chinese hamster ovary?CHO?cells and Ca²⁺luminescence bioassay,we tested the functional characterizations of BdAKHR and BdCrzR;Analyzed the spatial and temporal expression patterns of these four genes by qPCR and localization of BdAKH,BdCrz neurons by immunohistochemistry;Finally,we elucidated the physiology functions of AKH and Crz signaling systerms in B.dorsalis by RNA interfernece?RNAi?.The main results in this thesis are as follows:1 Characterization of AKH,Crz and their receptors from B.dorsalisBased on the B.dorsalis genome assembly datasets and transcriptome of central nervous system?CNS?,we identified and cloned the cDNAs that encode BdAKH and BdCrz and their receptors?BdAKHR and BdCrzR?by nested PCR.BdAKH and BdCrz precursors have conserved structures compared with other neuropeptides,which consist of?Signal peptide+Mature peptides+Precursor-related peptide?.One mature peptide was predicted from BdAKH and BdCrz,respectively.Mature BdAKH sequence is QLTFSPDWamide.While the[His⁷,Ser¹¹]-Crz isoform?QTFQYSHGWTSamide?was predicted from B.dorsalis that has never been reported before and it was identified as the 8^th subtype of Crz.Both BdAKHR and BdCrzR are the typical GPCRs with seven highly conserved transmembrane domains.Phylogenetic analysis indicates that BdAKHR was most related to Drosophlia melanogaster AKHR,and BdCrzR was as closely related to Musca domestica as it is to D.melanogaster CrzR.When expressed in CHO cells,BdAKHR showed concentration-depending Ca²⁺responses when activated with AKH peptide(the EC₅₀ of BdAKH is 19.3 nM),and BdCrzR exhibited a high sensitivity and selectivity for Crz(EC₅₀ of BdCrz is 52.5 nM).Therefore,BdAKH was set as model ligand for BdAKHR and BdCrz was set as model ligand for BdCrzR.The functional characterization of the AKH/AKHR and Crz/CrzR signal systems of B.dorsalis lays a foundation for the following physiological function analysis.2 Expression and localization of AKH,Crz and their receptors in B.dorsalisThe stage and tissue-specific expression patterns of BdAKH,BdCrzR and their receptores were analysized by qPCR.The transcripts of both BdAKH and BdAKHR were mainly detected in the larval and adult stages,and the expressions of BdAKH and BdAKHR were very low during the embryonic/egg and pupae stage,which indicates that AKH signaling system may play important roles in larval and adult stages of B.dorsalis.For the tissue-specific expression profiling of the adult,the mRNA was only abundance in the corpora cardiaca/corpus allatum?CC/CA?,and BdAKHR was highly concentrated in the fat body of both sexes,and at medium levels in the midgut.Immunochemical localization of BdAKH also showed the production of BdAKH in the CC/CA of the larvae.The transcripts of BdCrz and BdCrzR showed similar expression levels in all the developmental stages.Relatively high levels of BdCrz and BdCrzR were found in the larval stages,low in the egg and pupal stages,and then increased in the adult stage with a medium level.For the tissue-specific expression profiling of the larvae,the BdCrz transcript was only detected in the CNS,and BdCrzR mRNA was accumulated both in the epitracheal gland?EG?,containing the Inka cells,and also the CNS tissues.For the tissue-specific expression profiling of the adult,BdCrz was mainly expressed in the CNS,BdCrzR was high accumulated in the fat body as well as the CNS.Immunochemical localization confirmed the production of BdCrz in the central nervous system?CNS?,specifically by a group of three neurons in the dorso-lateral protocerebrum?DL?and eight pairs of lateral neurons in the ventral nerve cord?VNC?of the larvae,and also three pairs of adult Crz-immunoreactive neurons were observed in a cluster in the dorsolateral region of the protocerebrum?DLP?of the adult.These results showed that the Crz signal system has diversed physiological functions in the larvae adult stages of B.dorsalis.3 The physiological functions of AKH signaling systerm in B.dorsalisThe roles of AKH signaling systerm in larvae development,lipid accumulation,sexual behavior and flight duration of the adult were examined by RNAi bioassays.The results showed that BdAKH knockdown of larvae caused a delay in pupation.The pupation time of the AKH peptide-injected larvae advanced,and the pupa weight were significantly less than those in control group.These results showed that the physiology function of AKH signal system is closely involved in the larval development in B.dorsalis.RNAi assays were performed with dsRNA against BdAKHR in adult flies of both sexes under starvation,feeding or food rescure?starved+fed?conditions.As major results,the knockdown of this gene resulted in triacylglycerol?TAG?accumulation.With RNAi-males,we observed a severe decrease in their sexual courtship activity when starved,but there was a partial rescue in copulation when refed.Also in RNAi-males,the tethered-flight duration declined compared with the control group when starved,which is confirming the dependency on energy metabolism.In RNAi-females,the sexual behavior was not affected,but their fecundity was decreased.Our findings indicate an interesting role of AKHR in the sexual behavior of males specifically.The effects are associated with TAG accumulation.4 The physiological functions of Crz signaling systerm in B.dorsalisThe expression of BdCrz or BdCrzR was knocked down via RNAi to investigate its biological roles of Crz signaling systerm in the larval-pupal transition and pupariation as well as the mating behavior in the adult.Our results showed that BdCrzR knockdown caused a delay in larval-pupal transition and pupariation?72 h delay?,and this phenomenon agreed with a delayed expression of ecdysis-triggering hormone?ETH?,tyrosine hydroxylase?TH?and dopa-decarboxylase?DDC?genes.We speculate that BdCrzR-silencing blocked dopamine synthesis,resulting in the inhibition of pupariation and cuticular melanization.Based on the results that Crz is exclusively expressed in the CNS in the brain,we performed the head-ligation in 2-day-old 3^rd-instar larvae with the aim to stop the transportation of Crz from the head to the rest of the body.Finally,injection of Crz or ETH in head-ligated larvae could rescue the effects.These findings provide a new insight into the roles of Crz signaling pathway components in B.dorsalis and support an important role of CrzR in larval-pupal transition and pupariation behavior.For the adult,RNAi-mediated BdCrz knockdown lengthened the mating duration in males,and knockdown of BdCrz or BdCrzR strongly decreased male fertility in the following three days,while the courtship behavior and mating efficiency were not affected.The reduced number of sperm in the reproductive organs of mated females indicated that Crz knockdown in males reduced sperm transfer.These findings indicated that Crz contributes to the reproductive physiology of the oriental fruit fly B.dorsalis by regulating sperm transfer in male adults.In conclusion,we provided further understanding on the multiple functions of AKH/AKHR and Crz/CrzR in B.dorsalis.AKH signaling systerm modulates the larval development,mating behavior and flight ability of B.dorsalis through its energy metabolism function;Crz/CrzR plays important role in the larval-pupal transition and pupariation,which provided further insights into the machinery of insect pupariation.Furthermore,Crz signaling pathway also controls the reproduction of male adult via sperm transfer B.dorsalis.Therefore,our findings indicate that both AKH and Crz signaling pathways could be successful new targets of novel insect control agents.