Studies On The Responses Of Bactrocera Cucurbitae To High Temperature Stress And Its Moelcular Basis

Bactrocera cucurbitae?Coquillett?,i.e.melon fly,is a kind of important pest which has various fruit and vegetable hosts,wide distribution in tropical and subtropical regions.High-temperature can greatly affect the development and fecundity of the melon fly.For the evaluation of high-temperature effects of the melon fly.To evaluate the effects of high-temperature on adult melon flies,this research studied the response mechanism of adult melon fly under high temperature stress from multiple aspects,such as physiological,biochemical metabolism,transcriptional and proteomical.The main results are as follows.1?The effects of different high-temperature conditions on the survival,fecundity and behavior of the melon fly.Considering the characteristics of high-temperatures in the field,we set constant temperature tests?temperatures at 33?,37?,41 ? and 45?,respectively?and gradient temperature tests.In constant temperature tests,the melon fly was treated in 33 ?,37 ?,41? or 45 ? for 1h,2h and 3h.In long-term changing high temperatures test,the melon flies were treated in gradually increasing eight temperatures?temperatures raised from 31??33?,34?,35?,36?,37?,41?C and 45?in 7h?for once,twice or three times.The survival,fecundity,trend temperature,mating and oviposition behaviors of melon flies in different treatments were observed.The results showed that except for the 45 ?-1h treatment,high-temperature treatments were in general not favorable for melon fly's survial and fecundity.In the 45?-1h treatment,the total average eggs laid per female,the daily average eggs laid per female,mating and oviposition times were 692.7 eggs,20.4 eggs,41 times and 47 times,respectively,which were higher than in the control?666 eggs,9.6 eggs,38.3 times and 41.3 times,repectively?.Other temperature treatments showed with the increase of temperatures and duration,all the indexes declined.Trend to temperature behaviors of the melon fly were highly correlated with temperature and duration.It increased with increase of temperature,and declined with increase of duration.The highest trend to temperature were reached in the 45?-1h treatment,with females and males preferred 40.2? and 39.9? respectively.2?The impact of different high-emperature treatments on physiological metabolism in melonflies.Determination of six kinds of physiological metabolism index?include:Hydroxyproline,MDA,AChE,SOD,T-AOC and POD?in the melon fly after 1h of CK1?25 ??,37 ? and 45 ? treatments.The results showed that,With rising temperatures,SOD,AChE,MDA and Hydroxyproline activity were increased significantly than CK1,465.7?U/g FW??0.4??mol/min/gFW??74.0??mol/g FW?and 0.5??g/mL?were 45?-1h treatment respectively,the highest record among all treatments.while T-AOC and POD keep high stability high activity,were 55.3?FRAR Ag FW?and 31.2?U/mg protein?after 45?-1h treatment respectively.3.The development rate of ovaries dissection in the melonflies after the different high-temperature treatments.After 1h CKl?25 ??,37?and 45 ? high-temperature treatments,we found that as the rise of temperature,the overall fecundity and daily fecundity had been arising and reached peak after 45 ?-1h treatment,which were 648 eggs and 21.6 eggs respectivey.Ovarian development speeded up as the rise of temperature and was fastest after 45?-1h treatment.It only took 6d to grow mature and lay eggs compared with 12d of reference sample.4.High-throughput sequencing and analysis of transcriptomic and proteomic regulation responses of melonflies under different high-temperature conditions.After 1h CK1?25 ??,37?and 45 ? high-temperature treatments,conjoint analyses were carried at both transcriptional and translational levels.In the transcriptional levels,a total of 14,492,121,840 nt sequence were acquired and 46,826 Unigene assembled with total length of 72,693,277 nt.The result of genetic variations between different treatments shows that 39,914 unigene were up-regulated and 6,491 unigene were down-regulated at CKiVS 37?-1h;30,720 unigene were up-regulated and 15,434 unigene were down-regulated at CKi VS 45 ?-1h;8,668 unigene were up-regulated and 37,471 unigene were down-regulated at 37 ?-1h VS 45 ?-1h.In translational levels,405,232 spectrograms were got by protein quantification.After analyzing by the Mascot software,50,075 spectrograms?42,122 spectrograms unique?were matched and 3,184 proteins and 14,302 peptides?13,470 Unique peptides?were identificated.At CKiVS 37?-1h,141 sequences were up-regulated and 94 sequences were down-regulated;85 sequences were up-regulated and 83 sequences were down-regulated at CK1 VS 45?-1h;73 sequences were up-regulated and 182 sequences were down-regulated at 37?-1h VS 45?-1h.Based on this results,we selected seven target genes with different expression which are closely related to reproduction and thermal stress?Vg-1?Vg-2?Vg-3?Vitellogenin Receptor?JH-Inducible Protein?JH-Epoxide Hydrolase-2 and HSP70?.The results showed that as the temperature rose,three kinds of Vitellogenin?Vg-1?Vg-2 and Vg-3?and Vitellogenin Receptor responded differently and there were significant transcriptional level difference expression than CKi.Only the Vg-3 protein level showed a tendency to decrease.Both JH-Inducible and JH-Epoxide Hydrolase-2 had difference expression at both transcriptional and protein levels,in which the former showed a tendency to increase and the latter showed a tendency to decrease.Heat shock protein 70?HSP70?both at the transcriptional and protein levels,had difference expression at both transcriptional and protein levels.And with the rise of temperature,expression quantity has been increased,indicating that under high-temperature stress conditions,the start of the substantial expression of heat shock protein,protect the worms in response to high-temperature stress.5?qRT-PCR analysis of selection of reference genes and target gene in the melon fly after high-temperature stress.To select the suitable reference genes in the adult female the melonflies under the treatment of different high-temperature stress,we determined female adult of the melonflies the mRNA expression stability of five candidate reference genes?GAPDH,SD,?-TUB,ACT and RPL1S?after the treatment of temperature stress at CK1?25 ??,37? and 45 ? by qRT-PCR method.Then three software-based approaches?Bestkeeper?Normfinder and GeNorm?were used to analyze the expression stability of five candidate reference genes.The SD values of five reference genes stability by BestKeeper method in the ascending order were SD?0.06?=GAPDH?0.06?<RPL13?0.11?<?-TUB?0.15?<ACT?0.23?.The stability values of five reference genes stability by Normfinder method in the ascending order were SD?0.004?=RPL13?0.004?<GAPDH?0.005?<?-TUB?0.006?<ACT?0.010?;The M values of five reference genes stability by GeNorm method in the ascending order were SD?0.011?=RPL13?0.011?=GAPDH?0.011?<?-TUB?0.012?<ACT?0.016?.SD is the suitable reference gene.RPL13 and GAPDH is take second place.Target gene expression dynamics analysis by qRT-PCR method base on SD and RPL13 as reference genes.The results show that the selected seven gene dynamic expression were consistent with the high-throughput sequencing results.6?Clone analysis of objective genes.Using the cDNA as template,the analysis of cloning and the sequencing of target genes to select results showed that,the obtained sequence with the sequence alignment wereVg-1,Vg-2,Vg-3,Vitellogenin Receptor,JH-Inducible Protein,JH-Epoxide Hydrolase-2 and HSP70,which fragment similarity was 99%,99%,99%,100%,99%,99%and 99%,respectively.After different templates amplified by the NCBI sequence homology alignment,the most similar sequences were:melon fly predicted Vg-1?XM₀11186834.1,ident:99%?,melon fly predicted Vg-2?XM₀11184207.1,ident:99%?,melon fly predicted Vg-3?XM₀11186836.1,ident:98%?,melon fly predicted vitellogenin receptor?XM₀1179026.1,ident:99%?,melon fly predicted JH-induced protein?XM₀11197968.1,ident:99%?,melon fly predicted JH-epoxide hydrolase-2?XM₀11198165.1,ident:99%?and melon fly HSP70?KM112021.1,ident:97%?.Each sequence of the gene fragment had high similarity with the results of high-throughput sequencing in this paper and homologous sequences NCBI registered.Ttherefore,we can prove the sequencing results of this paper as credible,and the selected target gene fragment was needed.In further studies,its function can be verified by RNA interference etc.