| China has the largest citrus cultivation area as well as the total yield throughout the world.Citrus fruits rank the top in China.Citrus production in Brazil and America has been reported to decrease significantly in recent years due to the occurrence of Citrus Huanglongbing.Hence,the increasing citrus production in China has played a critical role in stabilizing world citrus industry.Chinese citrus fly,Bactrocera minax is an important pest in citrus producing areas of China.Due to global warming and the frequent trade in fruits and vegetables,the damage caused by this pest aggravates over time,which affects the sound development of citrus industry.B.minax has only one generation in a year along with a 6 month long pupal diapause.As a result it is still impossible to domesticate this fly thereby delimiting the basic and applied research.Thus,this study was carried out to explore the early-response genes and the related mechanisms that terminate pupal diapause by 20E treatments in B.minax.Then we investigated the lower development threshold and sum of effective temperatures of B.minax pupae in different geographical populations.In addition,the biological characteristics of the parasitoid of B.minax were studied.This study provides the theoretical basis and technical reserve for the adult monitoring and sustainable green control technologies of B.minax.The main results are as follows:1 Effect of temperature and photoperiod on the pupal diapause of B.mianxThe pupae of B.minax were exposed to different temperatures（15°C,20°C and 25°C）and photoperiods（L:D=16:8,L:D=12:12 and L:D=8:16）treatments to explore the pupal diapause characteristics of B.minax.The results showed that low temperature initiates pupal diapause of B.minax.The larval survival rate,pupation rate and eclosion rate of pupae exposed to low temperatures were significantly higher than that exposed to high temperature,but 50%individual eclosion time was up to 170 days.At the same temperature,there was no significant difference in larval survival rate,pupation rate and eclosion rate in different photoperiod treatment.Furthermore,about 3%to 5%of this population were non-diapause pupae and could directly develop into adults at 25°C.The natural low temperature in the wild promoted the larval pupation,adult emergence,and adult eclosion synchronization of B.minax.Furthermore,photoperiod had no effect on the pupal diapause termination of B.minax.2 Pupal diapause terminated by using 20-hydroxyecdysoneDifferent concentrations of 20E topical applied or injected into diapausing pupae to terminate pupal diapause of B.minax.Metamorphosis after 20E treatments were observed under a light microscope and the gene expression patterns of related genes were monitored by q RT-PCR.And then gene function was confirmed by RNA interference.The results showed that injection of 20E was more effective in terminating pupal diapause than topical application.The optimal dose of 20E needed for diapause termination was 0.2μg.Under this dose,the pupal eclosion rate was up to 70%.At 22℃,the matamophosis occurred about 7 days after 20E treatment and adult eclosed 40 days after treatment.Pupal diapause termination with 20E had no effect on adult reproduction of B.minax.QRT-PCR results showed that the expressions of ecr,foxo,broad and other early-response genes increased rapidly after 20E treatment.We speculate the underlying mechanism in pupal diapause termination of B.minax by 20E treatment.3 Determination of the lower development threshold and sum of effective temperatures of B.minax pupaeThe lower development threshold and sum of effective temperatures of B.minax pupae in different geographic population and diapause status were compared under different thermostatic conditions.The results showed that the lower development threshold of B.minax pupae in Hubei and Shannxi were 11.92°C and 11.72°C,respectively.The corresponding sums of effective temperatures were 492.45 day degrees and 519.15 day degrees,respectively.The lower development threshold of 20E treatment and natural diapause were lower than the diapause pupae,which were 6.46°C and 10.36°C,respectively.The corresponding sums of effective temperatures were 614.16 day degrees and 449.34 day degrees,respectively.Linear regression analysis showed that,the lower development threshold and sum of effective temperatures of natural termination diapause pupae were more accurate,which were suitable for prediction of the occurrence of B.minax adults.4 Monitoring and trapping research of B.minaxSperical traps were used to monitor the eclosion time of B.minax adult.Combined with the soil and air temperatures in Hubei and Shaanxi,and the lower development threshold of natural termination diapause of B.minax pupae,the sum of effective temperatures of B.minax pupae in the field was calculated.The control effect of the spherical trap was evaluated from three different perspectives:1)different citrus varieties,2)different suspending density and 3)different suspending time.The results show that the sphere trap can effectively monitor the occurrence of B.minax adults in the field.The sum of effective temperatures of B.minax pupae in Hubei was 288.27 days degrees（soil temperature）and 350.56 day degrees（air temperature）.While that of Shaanxi were 283.77days degrees（soil temperature）and 340.79 day degrees（air temperature）.In addition,spherical trap can trap and kill the male and female adults of B.minax effectively in the wild.It is recommended that hanging spherical trap in citrus orchard in mid-May,1 to 2times.5 Identification and biological study of the parasitoid of B.minaxMorphological and molecular identification and related biological studies of a parasitic wasp of B.minax was carried out.The male parasitic wasp had significantly longer tentacles than the female,while the lengths of other parts were shorter than the female.The body and fore wing length arrive significant level.According to the blast result of the COI gene,the sequence of the parasitoid was close to three other species of the Diachasmimorpha genera.The similarities were more the 90%.The biological study suggests that the parasite wasp laid eggs into the larvae of B.minax and emerged from the pupae in the following year.The eclosion rate of the parasitoid was lower.So far,this is the first record of this parasitoid in China.It is named as long tail Chinese citrus fly cocoon bee（Diachasmimorpha feijeni）because of its morphological and obligatory parasitism characteristics and has potentials to be incorporated into future B.minax biological control programs.6 Transcriptome Analysis of Diachasmimorpha feijeni and 20E treatmentA mixed transcriptome database of larvae,female and male adults of Diachasmimorpha feijeni was carried out by using Illumina sequencing technology.Furthermore,the effect of 20E on the diapausing parasitoids was investigated.The results showed that,the data size of the transcriptome was about 10.88 Gb.In total 48,331 unigenes were obtained,and the average length is about 1,031 bp.Among 19,216 unigenes,about39.76%were annotated.Among these,18,249 unigenes were annotated to Nr database,the closest species was Microplitis demolitor,and the E values of 50%unigenes were less than1e-50.The similarity of 72%unigenes was more than 60%,indicating a high quality of the transcriptomic sequencing and splicing.At 23°C,the eclosion time of B.minax and Diachasmimorpha feijeni were almost synchronized after 20E injection.In conclusion,this paper pinpoints the characteristic of pupal diapause in B.minax,which is obligatory diapause.We figure out the way to rapidly break pupal diapause of B.minax through 20E injection,which facilitate for the basic research of B.minax.Further,the early regulatory genes（ecr,foxo,broad）after 20E treatment were verified and the underlying molecular mechanism of pupal diapause termination was revealed.In addition,the lower development threshold and sum of effective temperatures of B.minax pupae were measured.And the control effect of spherical trap was evaluated in the field.What is more,the parasitoid of B.minax was identified and turned out to be Diachasmimorpha feijeni.The above results enriched our knowledge of B.minax and laid the foundation for further understanding the regulatory mechanism of pupal diapause,the monitoring and early warning,as well as the sustainable control techniques of B.minax. |
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