Identification And Functional Analysis Of Genes Coordinates With Puparium Tanning In The Oriental Fruit Fly,Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis(Hendel)has been considered to be one of the most important agricultural pests around the world.It mainly lays eggs into fruits,and then feeds on them by larvae.The normal physiological development of B.dorsalis must go through several moltings and two metamorphosis(larvae to pupae and pupae to adults),which accompanied with a series of complex morphological changes,physiological and biochemical reactions.The tanning reaction of newly formed epidermis is one of the most critical steps in these processes.In B.dorsalis,the newly formed white puparium completed tanning reaction(sclerotization and pigmentation)in a short time to protect their bodies and quickly adapt to the changeable living environment.Furthermore,the identification and excavation more functional genes for the specific developmental stages of pest are very important to develop environmentally friendly growth regulators.In this study,we mainly focused on the critical period of the transition from larval to pupal stage in B.dorsalis,identifying and verifying the pathways and functional genes that might play a key role in the process of puparium tanning.The main results are as follows: 1 The identification of puparium tanning pathway genes and analyze their expression pattern analysis in B.dorsalisWe obtained 74.62 GB of clean data for the expression profiles sequencing during pupariation in B.dorsalis.About 70.09%-74.44% of the data from each sample could be matched in the genome of the fruit fly,and the average GC content and Q30 of the sequencing data were 41.54% and 91.61% respectively.A total of 11,721 genes were obtained in the tested samples,of which 11,311(83.76% of the predicted genes)were successfully annotated in the genome of B.dorsalis.For those genes that failed to annotate in the genome(420)were defined as new genes.Further analysis of expression patterns showed that a large number of genes were differentially expressed during the pupariation of B.dorsalis.The GO,COG and KEGG analysis showed that oxidative phosphorylation pathway,metabolic detoxification pathway(P450),juvenile hormone synthesis,metabolism,signal and transport pathway,ecdysone synthesis and signal pathway,and chitin synthetic and degradation pathway were all significantly enriched,suggesting that they may play a key role in pupariation of B.dorsalis.Importantly,we also found the key pathway and genes(tyrosine metabolic pathway and CP genes)that related to insect cuticle tanning had also undergone tremendous changes during pupariation,indciating their key role in insect puparium tanning.Based on the genomic data of B.dorsalis,24 tyrosine metabolic pathway genes were identified.Then,the expression patterns of these genes were studied in the different developmental stages(eggs,early 1st instar larvae,late 1st instar larvae,early 2nd instar larvae,late 2nd instar larvae,early 3rd instar larvae,wandering stages,late wandering stages,white puparium,6 h puparium,12 h puparium,3 d pupa,5 d pupa,late pupa and newly emerged adults).The results showed that BdTH,BdDDC,BdEbony,BdADC,BdTan,BdMCO4,Bdyellow-c,Bdyellow-f and Bdyellow-f2 genes were significantly highly expressed during the critical period of puparium tanning.According to the quinone tanning theory of insects,we constructed the pathway of puparium tanning reaction in B.dorsalis: Tyrosine hydroxylase(BdTH)catalyzed tyrosine to synthesis DOPA;Most Dopa are catalyzed by dopamine decarboxylase(BdDDC)to produce dopamine;Then some Dopamine is catalyzed by BdMCO4,Yellow,Yellow-e and Yellow-h to form dopamine pigments(dark brown or black);Another part of dopamine is catalyzed by BdEbony,BdADC and BdTan to form NBAD,which is then catalyzed by BdMCO4 to form NBAD-quinone.The NBAD-quinone was further cross-linked with CPs to complete the sclerotization,while the puparium pigmentation was accomplished by NBAD-pigment(yellow or red-brown)and dopamine pigments.Further analysis showed the different tanning reactions between puparium and adult epidermis,and tanning process during the eclosion was mainly depended on NADA-quinone.2 The functional analysis of BdTH in the puparium tanning of B.dorsalisThe BdTH gene(the initial reaction enzyme and rate-limiting enzyme of the whole tanning pathway)was selected to carry out the further functional research.We designed the primers based on BdTH genome sequence and cloned the BdTH gene sequence from the epidermis of larvae of B.dorsalis by nested PCR.The length of the open reading frame was 1,737 bp,encoding 578 amino acid residues.The predicted molecular weight of the protein was 65.3 kDa,and the theoretical isoelectric point(pI)was 5.44.Multiple sequence alignment showed that BdTH had no signal peptide at N-terminal,but had conserved acidic region(PI = 3.78),serine site(ser32)and C-terminal.Phylogenetic analysis showed that BdTH sequence was closely related to fruit flies,and TH proteins of Diptera were clustered into one branch.Then,we used qPCR and semi-quantitative RT-PCR to analyze the temporal and spatial expression patterns of BdTH genes.The results showed that BdTH had high expression in the epidermis(EP)and central nervous system(CNS)tissues of larvae,and its expression level significantly increased during the transition from larvae to pupae stages,suggesting the importance of BdTH in puparium tanning reaction.The silence efficiency was about 50-60% after the injection of dsBdTH into the 5-day-old larvae,and the emergence rate of pupae decreased to 63%(94% in the control group).Similarly,when we injected or fed different concentrations of inhibitors(3-IT)to the larvae,the emergence rate of pupae significantly decreased in the high inhibitor concentration.In addition,when feeding TH inhibitor 3-IT with high concentration(10 mg/g),the puparium was pale(incomplete pigmentaion)and softer(incomplete sclerotization)than the control,and the pupal weight was significantly reduced as well.Scanning electron microscopy(SEM)showed that the pupa treated with the inhibitor had the abnormal phenotype of puparium,and the clear cross lines disappeared.We also found dopamine concentration significantly decreased after feeding inhibitors,but the downstream genes showed obvious feedback mechanism(the expression of BdDDC and BdEbony were significantly up-regulated).These results indicated that BdTH was essential for the tanning reaction of puparium of B.dorsalis.With the down-regulation of BdTH gene,the dopamine synthesis pathway was inhibited,which resulted in the significant inhibition of the tanning reaction of puparium.In this experiment,we also found that the production of melanin in 5-day-old larvae treated with inhibitor 3-IT was significantly lower than that in the control group,and the resistance to E.coli was significantly reduced,accompanied by a significant decrease in the expression of four antimicrobial peptide genes(Bddefensin,Bdattacin,Bdcecropin-A and Bdcecropin-B).These results showed that BdTH not only participated in the tanning reaction of puparium,but also played an important role in the immune response of larvae during pupariation in B.dorsalis.3 The functional analysis of BdDDC in the puparium tanning of B.dorsalisIn this study,the BdDDC gene was cloned from the epidermis of larvae by the nested PCR.The length of the open reading frame of the gene was 1,428 bp,and it could encode 475 amino acid residues.The predicted molecular weight and theoretical isoelectric point(pI)of the protein were 53.3 kDa and 6.29 respectively.The multiple sequence alignment showed that the DDC enzyme sequences were highly similar among insects.BdDDC was highly similar to D.melanogaster(82.53%),Bombyx Mori(72%)and Ceratitis capitata(74.53%).At the same time,the protein sequence of BdDDC contains glycosylation sites at N-terminal,pyridoxal-5-phosphate binding sites and a glycine-rich region.Similar to the results of BdTH,the phylogenetic tree shows that the DDC proteins of Diptera insects are clustered into one single branch as well.Then,we detected the expression patterns of BdDDC gene in different tissues of larvae of B.dorsalis by quantitative RT-PCR and semi-quantitative RT-PCR.The results showed that BdDDC was highly expressed in EP and CNS tissues of 3rd instar larvae.In addition,the expression of BdDDC gene significantly increased during the puparium tanning process.At the same time,we also detected the activity of BdDDC enzyme in different developmental stages.The results were consistent with the expression pattern of BdDDC gene.Therefore,we concluded that BdDDC may play an important role in pupariation and puparium tanning of B.dorsalis.After feeding DDC enzyme inhibitor to 5-day-old larvae,the BdDDC enzyme activity significantly decreased.The amount of dopamine significantly decreased,four key genes that involved in cuticle pigmentation(BdMCO4,Bdyellow-c,Bdyellow-f and Bdyellow-f2)were all up-regulated,the duration of pupariation was significantly delayed,a large number of black pupparium appeared,the pupal weight and emergence rate of B.dorsalis were significantly decreaed.The scanning electron microscopy(SEM)results showed that the pupa surface was irregular and the puparium thickness was significantly reduced.These results all confirmed that BdDDC was essential for the puparium tanning(sclerotization and pigmentaion)in B.dorsalis.4 Identification of CPs and Functional analysis of CPAP3 genes in pupariation of B.dorsalis 4.1 Identification of CPs and analysis of their expression patterns in B.dorsalisBased on the on the genome of B.dorsalis(NCBI Assembly: ASM78921v2),164 CP genes were identified by sequence alignment,which belong to 9 families,including 97 CPR,25 Tweedles,10 CPAP1,8 CPAP3,9 CPLCA,4 CPLCG,5 CPLCP,1 CPCFC and 5 CPF family genes.Further sequence analysis revealed that each family had its own conserved sequence: CPR family contained R&R motif;Tweedle protein had four conserved amino acid regions,region I contained a KX2Y/F motif,region II contained a KX4-5FIKAP motif,region III contained a TX2 YVL motif,region IV contained a KPEVYXFV/IKY motif,CPAP1 and CPAP3 protein had one and three chitin binding domains(ChtBD2)respectively;CPLCA protein contains conserved retinin domain;CPLCG protein sequence contains 35 conserved amino acid motifs,and there is a label motif G-X2-H-X-A-P-X2-G-H at the 9th-20 th position of the 35 sequences;CPLCP protein sequence contains high-density proline PV and PY repeats;CPF family has 44 amino acid residues;The final CPCFC protein sequence is a conserved region of 16 amino acids with 3 copies and ends with C-X5-C motif respectively.Then,we carried out phylogenetic analysis of CPs from D.melanogaster(model insects),C.capitata(closely related to B.dorsalis)and Lepidoptera,Hymenoptera,Coleoptera insects.The results indicated the unique CPs gene clusters and cuticle development patterns of Diptera insects.The present study also showed that some CP genes evolved independently among different species of insects,while others evolved based on their specific conservative domains.The expression patterns of CP genes in different tissues of B.dorsalis were analysed,and most of the CP genes could be detected in the cuticle of fruit fly.We also found that some specific CP genes could be dected in the internal organs of B.dorsalis,such as fat body,gut,ovary,testis tissues,suggesting the important and diversity functions of CPs.Importantly,the sequence of CPAP3 family genes of B.dorsalis are conservative among insects,and had high expression in the cuticle of 3rd instar larva(pre-pupation),indicating their important roles in the formation of puparium in B.dorsalis.4.2 Functional analysis of CPAP3 family genes in pupariation of B.dorsaslisCPAP3 family proteins of B.dorsalis contain three conserved chitin binding domains,impling their potential chitin binding capacity.Then we selected CPAP3-D2 and E proteins to verify.Firstly,the purified recombinant protein was obtained by prokaryotic expression,and then the protein was incubated with chitin resin.The binding efficiency of CPAP3-D2 and CPAP3-E proteins to chitin was 66.72% and 52.78%,respectively,which was significantly higher than that of bovine serum protein(BSA)by 18.06%.Spatiotemporal expression patterns of CPAP3 family genes showed that CPAP3-A1,B,E and E2 genes were highly expressed not only in the cuticle of 3rd instar larva,but also in the critical period of pupariation,suggesting these four genes minght play a key role in the puparium formation.Subsequently,we also found that the gene expression of CPAP3-A1,B,E and E2 were all significantly down-regulated after 12 and 24 hours of juvenile hormone treatment,whereas those genes were significantly induced after 20 E treatment.We used RNAi technology to explore the possible functions of CPAP3-A,B,E and E2 genes in the pupariation of B.dorsalis.When the dsRNAs of these genes were injected into 5-day-old larvae,the silencing efficiency of these genes would be maintained in the range from 35% to 60%,and the pupariation time would be significantly prolonged with the delay of larval development.Moreover,when we reduce the expression of BdCPAP-E gene by RNAi,40% of the larvae died(the body becomes stiff and tense and lacks relaxation).Additionally,the expression of CPAP3-D2 gene in eggs was significantly higher than that in any other developmental stages.The results of immunohistochemical experiments on frozen sections of eggs showed that CPAP3-D2 was an important structural protein in the eggshell of B.dorsalis.The expression of CPAP3-D2 gene in the ovaries of was significantly higher in mature ovary.Then,when we injected dsCPAP3-D2 into the female adults,the ovarian development and the formation of egg flaps were significantly affected.This study demonstrates that CPs were not only involved in puparition,but also essential for their reproductive development.In conclusion,the present dissertation focused on the critical period of puparition in B.dorsalis.On the basis of the puparium tanning pathway of B.dorsalis,we targeted the key genes of BdTH,BdDDC and CPAP3 respectively,and explored their important functions in the tanning reaction of puparium.These results not only greatly deepened people's understanding of tanning reaction in B.dorsalis,but also screened out many functional genes,which have potential to become new targets for pest control.