Mechanism Of Summer Diapause Induction Of Delia Antiqua(Diptera:Anthomyiidae)

The onion maggot,Delia antiqua?Diptera:Anthomyiidae?,is a worldwide distributed liliaceous pest.Onion maggot is one of the insect species that with characteristics of summer-and winter diapause,and has strong environmental adaptability.Onion maggots can be reared in large scale with artificial feed in laboratory.Meanwhile,they are phylogenetically related to Drosophila melanogaster,so it can be used as an ideal model for comparative studies on winter,summer and non diapause.Diapause is an adaptive developmental plasticity of crucial ecological importance.Current research on diapause in onion maggot mainly focus on the biology,ecology,physiology and morphology and molecular mechanisms comparisons between diapause and non diapause individuals,genetic and environmental control of diapause termination,so far the molecular regulation of the most“upstream”diapause induction stage remains unclear.Take the onion maggot as research material,the optimal environmental conditions of diapause induction were determined by combination of different photoperiod and temperature.Effects of temperature and photoperiod on diapause induction were ascertained.Transcriptome dynamic changes of sensitive stages between non-and summer diapause induction were compared.Genes potential related to summer diapause induction were screened through pathway analysis.Expression pattern and potential roles in regulation of diapause induction of several genes were analyzed by qRT-PCR.The main results are listed as follows:?1?The model system of comparative research on diapause and non diapause was established with the model species of onion maggot.The optimal condition of diapause induction and effects of photoperiod and temperature were determined by combination of different temperatures and photoperiods.Under 16,18 and 20?,diapause incidence increased significantly with the shortening of day length.Diapause incidence is low under LD16:8 and low temperature condition.Diapause incidence increased gradually with the decreasing daylength.The vast majority of individuals do not enter diapause in the narrow range of daylength?16h daylength?.All or most of the individuals entered diapause under the condition of daylength>10h,22,24 and 26?,and daylength<12h,16,18 and 20?.Diapause rate under 22,24and 26?had no significant difference when daylength>16h.Diapause rate decreased when daylength<12h with the temperature rising.Effects of temperature on diapause induction of onion maggot accompanied with the photoperiod effect.Effect of temperature on diapause induction can be shown only when the daylength<16h.Diapause of the onion maggot belongs to a typical long day-short day type.Photoperiod effect plays a main role in winter diapause induction,and is accompanied with the temperature effect,while,summer diapause induction is mainly controlled by the temperature.?2?Three instars of the larvae and the sensitive stage of diapause induction were determined.According to the variation of the cephalopharyngeal skeleton and the mouth hook length and the changes of the morphological characteristics of anterior spiracle and anal spiracle,the larval stage is divided into three instars.The most sensitive stage of diapause induction was determined by using diapause inducing condition 26?,LD16:8 and diapause averting condition 20?,LD16:8.Eggs and larvae reared under 26?,LD16:8 were transferred to 20?,LD16:8 day by day.At the same time,eggs and larvae reared under 20?,LD16:8 were transferred to 26?,LD16:8 day by day.According to the pupal diapause rate in each group,the most sensitive period of summer diapause and non diapause larvae were 9d and 4d of the3^rd instar,respectively.This will provide a basis for accurate sampling points for RNA sequencing.?3?Here we used high-throughput RNA-Seq to identify candidate genes and processes linked to summer diapause induction by comparing the transcriptome differences between the most sensitive larval developmental stage of summer-diapause?SD?and non-diapause?ND?.The results showed that the differentially expressed genes mainly belong to different structures,regulation,metabolism and transporter proteins.The possible regulators of diapause induction are related to circadian rhythm,thermosensitive and immune defense.The expression patterns of hsps and circadian clock genes suggest that they play an important role in the summer diapause induction of the onion maggots.1)Larvae were collected at 2:00,10:00 and 18:00 of 9-day-old and 4-day-old3rd instar larvae rearing under 20±0.5?,LD16:8 and 26±0.5?,LD16:8,respectively.Six cDNA sequencing libraries of summer diapause and non diapause induction sensitive stage larvae were constructed,and the libraries were sequenced using HiSeq2000 technology.The reference sequence of the transcriptome was reassembled to obtain 59,389 UniGene,with an average length of 1392bp.These unigenes were compared with each protein database.A total of 16,641 unigenes were divided into 65 GO functional groups.In GO functional groups,cellular processes and single-organism processes are dominant in biological process groups.Most of the 21 components of the cell composition are cell and cell part.In the molecular function group,most genes belong to the binding and catalytic activity.2)Based on GO annotation,the differentially expressed genes were assigned to different pathways.Several functional terms related to lipid,carbohydrate and energy metabolism,environmental adaption,immune response and aging were enriched during the most sensitive SD induction period.Some of the unigenes in the metabolism pathway are related to fat metabolism,stress response,oxidative stress protection,immune response and circadian rhythm.Among them,the heat shock proteins are representative in the down-regulated genes,and the circadian clock genes play important role in insect diapause regulation.3)The expression and regulation of circadian rhythm,life regualtion,stress response,oxidative phosphorylation,fatty acid metabolism,TCA cycle and MAPK signaling pathway related genes were differentially expressed in ND.It indicates that the metabolism transformation of fat,carbohydrates and energy accumulation occured during the diapause induction stage.These metabolism pathways play an important role in the growth and diapause induction of onion maggots.While genes related to steroidogenesis,fatty acid metabolism,the citric acid cycle,oxidative phosphorylation and stress response showed circadian expression patterns indicated circadian clock involved in diapause induction of the onion maggots.Subsequently,10 representative genes with significant differential expression were verified by quantitative PCR,which proved the reliability of sequencing and expression analysis.4)Circadian and diapause induction related expression patterns of hsp23,period,timeless,casein kinase1,shaggy and takeout were analyzed.Differential regulation of hsp23 during diapause induction and diapause stage maybe related to response to thermal injury inflicted on larvae and diapausing pupae.Differential expressed patterns between non diapause and summer diapause induction larvae suggest the potential relationship between circadian clock genes and summer diapause induction in Delia antiqua.5)By using PPI network analysis and iRefIndex database to identify the genes co-expression network,gene modules and other closely related genes by gene network analysis to find the possible regulating components that induce summer diapause.Results showed that the genes deltex,hsp23,Hsp60,hsp83,Jra,COX2,period,mastermind and shaggy may play an important role in the onion maggot summer diapause induction.6)The gene expression patterns of six time points in the most sensitive period of non diapause and summer diapause induction were analyzed by RT-qPCR.The target genes timeless,period,Casein kinase1,takeout,and shaggy were expressed in circadian oscillation patterns.Timeless,CK1 and period showed significantly diapause induction-related up-regulation,and shaggy showed significantly diapause induction-related down-regulation.In S2 vs.N2,takeout is significantly down-regulated.However,it is significantly up-regulated in S10 vs.N10 and S18 vs.N18.Hsp23 was down-regulated during the sensitive stage of summer diapause induction,when compared with non-diapause induction,and up-regulated following entry into diapause.The results indicate differential regulation of hsp23 during diapause induction and diapause stage maybe related to response to thermal injury inflicted on larvae and diapausing pupae.In conclusion,diapause induction in onion maggots is an important upstream physiological and ecological stage during its development.It is a complex dynamic physiological process.There are multiple molecular pathways and genes involved,which are regulated by extremely complex hormones and environmental regulation mechanisms.In this study,we constructed the first transcriptome and expression database of the diapause induction sensitive period of the species.We identified the genes related to onion maggots diapause induction.This study enriches the study of insect diapause and lays a foundation for understanding of the molecular mechanism of insect diapause and its regulation.