| The Chinese white wax insect is a kind of important resource insect,and white wax,the secondary metabolite secreted by male larvae,is one kind of natural macromolecule compound and has been widely used in medicine,food and other industries for thousands years.Chinese white wax insect is typical of sexually dimorphic: the female undergo hemimetabolism,and the male undergo holometabolism.So it is great significant species for studying the evolution and metamorphosis of insects.In order to elucidate the regulation and effect of juvenile(JH)and ecdysing hormone(20E)in the development of white wax insect,the morphological characteristics and dynamic regularity of juvenile hormone and ecdysone titers in different development stages of male and female insects were firstly studied in this paper.Then,the differentially expressed genes of the transcriptome of the 2nd instar female and male insects were analyzed,and JH and 20E-related genes expression level in each development stage were measured by real-time quantitative PCR(q RT-PCR).Finally,we also analyzed the impact of the spraying of exogenous juvenile hormone analogues(JHA)on the early stage of 2nd instar male larvae and on the expression of JH and 20E-related genes.The main results are as follows:1.Dynamic changes of hormone titersJH and 20 E titer of male and female insects were determined by high performance liquid chromatography(HPLC)and enzyme linked immunosorbent assay(ELISA),respectively.The results showed that JH titer are higher in females than males(higher in hemimetabolism than holometabolism);During middle stage of 1st instar,as the starting point for the development of sexually dimorphic,high concentration JH(91.640 pg/mg)induced hemimetabolism of females,and low concentration JH(8.980 pg/mg)caused holometabolism in males.During the transition period of development in white wax insects,when the difference between the 20 E and JH titers reach a certain value,and the development can be started at the new age.While differences of JH and 20 E was between 31.019 and 59.366 pg/mg,females and males can development normally.In the period of adult insect,20 E titer was relative higher than JH titer in both females and males.2.Study on the expression patterns of key genes related to endogenous hormone signaling pathwayThe expression patterns of key genes related to the endogenous hormone signaling pathways were detected by q RT-PCR technique in male and female,and were combined with the changes of hormone titers for further analysis.The results suggested that FPS,FDS,PMVK,JHE and JHEH involving in the JH synthesis pathway were observed high expression only at 2nd and adult stages of females.JHAMT and FAMe T were highly expressed only at 2nd instar of males.Therefore,FPS,FDS,PMVK,JHAMT,FAMe T,JHE and JHEH are possible the putative key genes regulating the development of the sexually dimorphic of white wax insect.From the larvae to adult stage,the expression of JH synthesis and degrading enzyme genes were increased gradually in females.The expression level of 20 E synthesis gene in adult stage was higher than that in larva stage in females.And in males the highest expression level of those genes peaked at the true pupal stage.The expression trend(the level of expression in adult stage was higher than that in larva stage)of Me T gene,encoding JH receptor protein,was opposite to the change of JH titer in females,and its expression trend in males(larva stage was higher than adults)was basically consistent with the change of JH titer.However,the expression changes of Ec R,encoding ecdysone receptor protein,were consistent with the changes in 20 E titer in females and males.The results indicated that the receptor protein Me T was regulate negatively by JH in females,but positively in males.While 20 E positively regulate the expression of Ec R gene encoding its receptor protein in both females and males endogenous hormone signaling pathway.3.Effects of exogenous hormones on the instar development and the expression of genes invloved in hormone signaling pathwayThree concentrations of exogenous hormones JH1(1.25mg/ml),JH2(2.5mg/ml)and JH3(25mg/ml)were used to treat 2nd-instar males by spraying,smearing stem and injection,and then the excreted wax amount,eclosion rate,body length and weight were measured,while expression patterns of genes related to hormone signaling pathway were analyzed.The results showed that the individuality wax secretion amount reached up to 41.50% in the Kunming city,and 25.56% in the Emei city after spraying medium concentration of JHA(2.5mg/ml).The eclosion rate of pupae reached 62.70%~81.62% under three concentration and treatment methods,and the promoting effect of painting is better than spraying and injection at the same concentration.while promoting the emergence of pupae,JH3 significantly inhibited the development of body length and body weight.JH2 up-regulated the expression of the JH-related genes and Ec R,and JH3 down-regulated the expression of Me T and Ec R.These results indicated that JHA could regulate the expression of genes involving in hormone signaling pathway.And JH2 may shorten the time to reach the peak of wax secretion or prolong the development of the 2nd-instar males so that increased individuality wax secretion amount in Chinese white wax insects. |
PDF Full Text Request