| The Neoseiulus barkeri is a natural predator of many sucking insect in agriculture.It widely reckons as one of the best biological control products,for its large amount of predation,rapid propagation and short development period,and it occupies a broad application area in predatory natural enemy products in China and abroad.The survival in winter is crucial to N.barkeri,owing to the high mortality in this time.However,no relative research was found in the winter survival of N.barkeri.Therefore,we assessed the effects of temperature and photoperiod(L:D)to the diapause of this mite,and hope to determine the optimal conditions for its efficient survival.Moreover,genes,associated with diapause of mite,were screened by transcriptome analysis.And we also revealed the change pattern of the Vg and VgR at different developmental stages.Our research is aim to uncover the mechanism of diapause in N.barkeri.For the key roles of the production and uptake of yolk protein in the propagation of oviparous organisms,understanding the molecular regulation of Vg and VgR will contribute to the control of its reproductive principles.Eventually,it will improve the biological control of beneficial mites,and control the occurrence of harmful mites.Our main results are shown as following:1.The impacts of temperature and photoperiod to the development of N.barkeri:N.barkeri were rear to adult with the same combination of temperature(14℃,16℃,18℃,20℃ and 22℃)and photoperiod(0 h,8 h,16 h and 24h).N.Barkeri eggs only developed nymph mites in 14 ℃,whereas other temperatures maintained the completed developmental process.The photoperiod photoperiod could result in a faster development process then a shorter process,under the same temperature.Moreover,temperature influenced the mite development significantly with the same photoperiod,which shown as the higher the temperature,the faster the development.The whole development process lasted 33 d in 16℃,while it shorten to 13 d in 22℃.Therefore,temperature and photoperiod are the crucial ecological factors,which affect the development process of N.barkeri.2.The impacts of temperature and illumination on the rates of diapause and survival of N.barkeri:The critical photoperiod of diapause is a condition with 18℃ and 19 h photoperiod.Whatever the duration of photoperiod,the female adult of mite will enter the diapause state in 16℃,but the survival rate will less than 10%.In addition,under 18℃,the N.barkeri showed a decreasing trend of diapause rate with the longer photoperiod,but the survival rate improved.The rates of diapause and survival both remained higher when L: D equal to 8:16.The survival rate of female mite adult was 83% when the temperature higher than 20℃.The female adult of mite displayed a very low diapause rate in 20℃,and it will not enter the diapause stage in 22℃,regardless of the photoperiod duration.3.The impacts of temperature and photoperiod to the starvation tolerance of N.barkeri.:N.barkeri were rear to adult with the same combination of temperature(14℃,16℃,18℃,20℃ and 22℃)and photoperiod(0 h,8 h,16 h and 24h).There were significant differences in the resistance time between female adult mites.N.barkeri female adult mites in the case of 18 ° C L: D = 8: 16,the survival time of the starving instars was the longest(21 ± 0.29 days).Under the conditions of 18 ℃ L: D = 8: 16,the diapause is higher,the survival rate is higher,the survival time of the starving instars is longest.Comprehensive consideration of diapause,survival and resistance to hunger,it’s the best condition for the diapause of the N.barkeri.4.Transcriptional analysis of diapause mites of N.barkeri.This study analyzed and established the transcriptome sequencing of diapause mites of N.barkeri by the high throughput sequencing platform,and assembled the sequencing reads into Unigene using the de novo strategy,and conducted annotation of GO functional classification and KEGG pathway analysis to the unigene.There were 55013 transcripts and 23144 genes reached 25478066 bp.The average length of the gene in the spliced gene was 1100 bp,the longest was 21289 bp and the N50 value was 2114 bp,which showed patchwork genes quality was better.The 23144 Unigenewere annotated using the blast tools,7863 of which were annotated to Swiss-Prot,8480 toPfam,12625 to NR,8004 to KOG,6225 to KEGG and 7192 to GO.The functions of 7192 to GO were transcription,translation,transcriptional regulation,autophagy,cell division and signal transduction and so on.The functions of 6225 to KEGG were organismal metabolism,systems,environmental information processing,genetic information processing and cellular processes.Our transcriptome study on diapause mites of Neoseiulus barkeri obtained a lot of genetic data,Which laid the foundation for further study of diapause from two levels of gene and protein.5.Sequence analysis of Vg1,Vg2 and VgR in N.barkeri.Using relative phytoseiid mites Vg and VgR as the reference sequence,we screened two conventional Vg and one VgR from N.barkeri transcriptome by local blast.The complete nucleotide sequence of NbVg1 is 5623 bp with a 5568 bp of open reading frame,which encodes a 212.52 kDa protein of 1856 amino acids including a putative signal peptide of 16 residues.The calculated isoelectric point was 8.57.Containing 238 basic aa,224 acidic aa,582 hydrophobic aa,539 polar aa,13 glycosylation sites(N-X-S or N-X-T).NbVg1 comprises three conserved regions including Vitellogenin-N domain(Vit-N),DUF1943 and von Willebrand factor type D domain(vWD),and polyserine domains a RXXR cleavage signal(RYAR)and highly conserved GL/ICG and DGXR motif.The complete nucleotide sequence of Nb Vg2 is 5795 bp with a 5532 bp of open reading frame,which encodes a 211.24 kDa protein of 1843 amino acids including a putative signal peptide of 16 residues.The calculated isoelectric point was 8.98.Containing 235 basic aa,209 acidic aa,556 hydrophobic aa,550 polar aa,8 glycosylation sites(N-X-S or N-X-T).NbVg2 comprises three conserved regions including Vitellogenin-N domain(Vit-N),DUF1943 and von Willebrand factor type D domain(vWD),and polyserine domains a RXXR cleavage signal(RYAR)and highly conserved GL/ICG and DGXR motif.The complete nucleotide sequence of NbVgR is 5335 bp with a 5109 bp of open reading frame,which encodes a 191.14 kDa protein of 1702 amino acids containing a total of 198 basic aa,255 acidic aa,450 hydrophobic aa,508 polar aa,8 glycosylation sites(N-X-S or N-X-T)including a putative signal peptide of 19 residues.The calculated isoelectric point was 5.53.The VgR belongs tolow density lipoprotein receptor family.As a result,the NbVgR has four typical conserved domains,including ligand-binding domain(LBD),EGF-precursor homology domain(EGFP),transmembrane domain and cytoplasmic domain.NbVgR has two LBDs,each containing four and eight class A repeating domains,and has two EGF-precursor homology domain,each contain four EGF-like repeating domains.6.Expression analysis of NbVg1,NbVg2 and NbVgR in N.barkeri:The transcriptional expression levels of NbVg1,NbVg2 and NbVgR were detected in all life stages(larva,nymph,pre-oviposition,oviposition,diapause and male adult).On development of N.barkeri,the qPCR results indicated that NbVg1、NbVg2 were detected very little transcriptional level in the larva of N.barkeri.The expression levels of NbVg1,NbVg2 and NbVgR increased in succession,and the levels of transcription of these geges were increased rapidly.The expression levels of NbVg1 and NbVg2 were the highest in the diapause period,and the expression level of NbVgR was the highest in the pre-oviposition stage,and the expression level of the oviposition period was the second.In addition,we detected low transcriptional level in the male of N.barkeri.Analysis show,ed that the expression levels of NbVg1 and NbVg2 in diapause were higher than those in other period,but the expression level of NbVgR in the diapause period was significantly lower than that of pre-oviposition and oviposition period.The expression level of NbVgR in diapause was significantly lower than that in undiapause female,which resulted in the lack of adequate intake of Vg in oocytes,which resulted in diapause and reproductive cessation of adult females. |
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