| Bemisia tabaci Mediterranean (MED, formerly referred to as the "Q biotype") was first found in Kunming of Yunnan province in China since2003, then spread widely and established population quickly with extreme temperature adaptability, stronger resistance to pesticides etc.. The MED whitefly replaced Bemisia tabaci Middle East-Asia Minor1(MEAM1, formerly referred to as the "B biotype") gradually after2007and had been the principal invasive species at some regions in our country, resulting in strong damage to crops. Previous researches discovered that the MEAM1whitefly indirectly benefits from the interactions with begomovirus via inhibited host plant defense responses, accelerated ovarian development and increased fecundity, then replaced the indigenous Asia113(formerly referred to as the "ZHJ1biotype") with a large number of offspring. Therefore, the competition among cryptic species of Bemisia tabaci may mainly result from different reproduction regulation ability. Study the molecular mechanism of reproductive regulation will not only provide a better understanding of the mechanism of Bemisia tabacfs reproduction related to quick population establishment, diffusion and outbreak, but also provide insights into the mutualistic mechanism with a plant begomovirus.As an important developmental stage in insect’s reproduction, the dynamic of vitellogenesis and its endocrine regulation mechanism are very important points need to be revealed. To confirm the hormone regulation pattern of vitellogenesis in MED, the full length of genes on hormone metabolism pathways were cloned, the dynamic of genes transcription and ovarian development after exogenous hormone treatment and eating different sugars were studied. In addition, the knock down effection of juvenile hormone esterase gene on vitellogenesis of MED was discussed. The main results are as follows:1. Molecular cloning, sequence analysis and developmental expression of genes related to endocrine regulation of MED reproductionThe cDNA sequences of MED vitellogenin receptor (ygr), juvenile hormone esterase(Btjhe), juvenile hormone binding protein (jhbp), ecdysone receptor (EcR) and ecdysone-induced protein75(e75) were cloned and blasted with other insect homologous proteins. The relative expression levels were detected on different developmental stages by real-time quantitative PCR. The results showed that:the cloned cDNA sequences of vgr, Btjhe, jhbp, EcR and e75were accurate and had the same conservative structure domains with other homologous proteins. The expression patterns of vgr, Btjhe and jhbp were consistent with that of vg, as the mRNA transcription was rising in the first, then reducing, and reached the highest on the eleven day after eclosion. It suggest the regulatory factor genes Btjhe and jhbp have a corelation with vg and. vgr genes, regulating the vitellogenesis and oogenesis of MED, while genes on the ecdysone pathway may either not participate in regulation or had no significant effects.2. The knock down effect of Btjhe on vitellogenesis of Bemisia tabacl MEDThe newly emerged MED females were collected and fed with the artificial feeding liquid incorporate synthesised Btjhe-dsRNA. Then the Btjhe expression level after various feeding time with1μg/μL and2μg/μL dsRNA treatments were detected. The results showed when eating with72hours on two dsRNA concentrations, the target gene was silenced by60%to90%. Through detecting the dynamics about ovarian development and related genes transcription levels while Btjhe gene was silenced, the relationship between juvenile hormone and the vitellogenesis of MED was explored. The results showed:when MED eating72hours with2μg/μL dsRNA in the feeding liquid, Btjhe gene was silenced by87%, and genes related to vitellogenesis were significantly down-regulated. The relative expression levels of vg, vgr and jhbp were down-regulated by74%,73%and86%respectively. It indicated silencing of Btjhe inhibited the expression of vg and vgr significantly. These results suggest JHE regulate vgand vgr expression in MED positively, and high expression level of Btjhe may promote vitellogenesis of MED.3. The influence of exogenous juvenile hormone treatment on the reproduction of Bemisia tabaci MEDPseudo-pupas with red eyes were treated with exogenous JH Ⅲ. Then the newly emerged females were collected for the detection of dynamics about ovarian development and gene expression of vg vgr, Btjhe and jhbp. Results showed that:application of exogenous JH Ⅲ up-regulated the expression of Btjhe, vg, vgr and jhbp by1.90to5.48fold,1.16to1.60fold,1.13to1.90fold and1.11to1.19fold respectively compared to the control. The number of the4rd grade eggs and ovarioles of JH Ⅲ treated MED reached the peak value at the first day after eclosion, which was2days earlier than contol MED. These results suggest:enhanced transcription level of Btjhe, which was stimulated by exogenous JH Ⅲ, promoted the specific degradation of JH and induced more gene expression of vg in MED. Exogenous JH also accelerated ovarian development in MED, induced the advanced eggs maturation. All these facts suggest JH and molecules on its metabolic pathway were important factors in regulating Vg synthesis of MED, and the vitellogenesis in MED was mainly regulated by JH.4. The influence of feeding with different sugars on the reproduction of Bemisia tabaci MEDNewly emerged adults were fed with artificial feeding liquid containing sucrose, fructose and glucose respectively. Then the dynamics of ovarian development and gene expression of vg vgr and Btjhe were detected on each day. Results showed:MED feeding with sugars had higher vg, vgr and Btjhe expression levels than control, and females feeding with sucrose had the highest transcription levels. Only females feeding with sucrose or glucose would have the biggest number of the4rd grade eggs and ovarioles, and reached maximum ovarian development for1to2days earlier than fructose. The number of the4rd grade eggs of females feeding with sucrose was significantly higher than fructose except the4and7days after eclosion. It suggest:sucrose feeding accelerated whiteflies ovarian development and egg maturation. So sucrose can be used in the combination with hormone treatment and RNA interference to study nutrition and hormone cross-talk on whiteflies’reproduction regulation. |
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