Vitellogenesis And Sequences Analysis Of Vitellogenin And Vitellogenin Receptor Gene In Whiteflies Bemisia Tabaci Gennadius (Hemiptera: Aleyrodidae)

The catastrophic outbreak of biological invasions had made scientists all over the worlds pay attentions to this problem. From the previouse researches, the mechanisms for successful invasion of whiteflies Bemisia tabaci are as follows:first, the asymmetric mating interactions; second, the vector-virus mutualism; third, the resistance of chemical pesticide. However, the physical and molecular mechanisms induced these ecological phenomenon were still poorly understood. It is important for us to verify these mechanisms and find an effective method for the management of this world pest. In our research, the reproductive behaviors in types of whiteflies were observed, the cDNA of vitellogenin and vitellogenin receptor were cloned, the characteristics of genes were compared and the developmental model was observed using RT-PCR. Examination of the difference among three biotypes of whiteflies was aimed to find the molecular and physiological mechanisms of successful invasion. The results are as follows:1 The morphology of reproductive system in whiteflies and comparison of the development of ovariesThe reproductive system of female is mainly composed of a pair of ovary, a spermatheca of 20μm, a pair of lateral oviduct and common oviduct. The ovaries of the whitefly were composed of 12-22 telotrophic ovarioles. Four developmental stages of ovary were divided according to lifespan of whiteflies. The oocytes in ovary were divided into four types, typeⅠ,Ⅱ,ⅢandⅣ. The morphology of male reproductive system was composed of a pair of tesis, male accessory gland and vas deferens. Compared with those ZHJ1 biotype whiteflies, no differences were examined on the development of ovary in the B biotype whiteflies fed on cotton plants.2 The oogenesis and spermiogenesis of Bemisia tabaciThe oogenesis of Bemisia tabaci was observed at the pupal periods. The oogonial stem cell differentiated into oocytes, nursecells and follicle cells during the formation of the ovaries. We detected ovarioles in females during pupal period. The spermatheca could be examined 48 h after eclosion. Four stages during oogenesis could be observed,1:trophocyte stage; 2: previtellogenesis stage; 3:vitellogenesis stage; 4:mature stage. The nutrition was transport to oocytes by receptor mediated way and transferred by the trophocytes. Only one oocyte was examined during oogenesis in ovariole. Sperms were differentiated by the sperm stem cell and the spermiogenesis could be divided into four developmental stages, each stage has their own structure and morphology was different.3 Preparation of the monoclonal and polyclonal antibody of vitellin (Vt) in B. tabaci.In order to study vitellogenesis and its endocrine regulation, we used hybridoma techniques, and developed monoclonal antibodies to Bemisia tabaci egg soluble yolk proteins. The monoclonal antibodie have an IgGl heavy chain and aκlight chain.ELISA was used to determine the titer of antibodies. The results revealed that monoclonal antibodies have a titer of 1:100000 and the polyclonal antibodies were 1:68000. The high specificity and affinity not only to the ovarian vitellin (Vt), but also to female hemolymph vitellogenin (Vg). Western-blot revealed that the two types of antibody had immunological reaction with the others whiteflies Vt and Vg. However, they had no immunological reaction with the male substances.4 The vitellogenesis and characterization of vitellogenin in B. tabaciWith a combination of gel chromatography and ion exchange column, the vitellogenin was purified from the soluble proteins fractions of crude egg extracts. And then, Native-PAGE and SDS-PAGE were applied to analysis the molecular composition of Vg protein, we found that the yolk protein had a native molecular mass of 380 kilodaltons and was composed of two similar subunits with molecular mass of 190 kilodaltons. Carbohydrates, lipids, and phosphorus were found associated with Vt as revealed by the respective stain ability to periodic acid-Schiff's reagent, Sudan Black B and Methyl Green Solution after gel electrophoresis. We realized that vitellogenin in B. tabaci belonged to phosphoric-glycosylated proteins. We also detected the Vg level in B. tabaci of different developmental stages using the method of sandwich ELISA. It was examined that the vitellogenin in B. tabaci was synthesized in pretended pupal stage and the level of Vg in hemolymph is decreased after eclosion. Increased Vg level was observed in the lateral stage. While the level of Vt in ovary kept increase after eclosion.5 cDNA cloning and expression analysis of vitellogenin in B. tabaciThe cDNA sequence of vitellogenin was cloned from B biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci is 6731 bp with 6474 bp of open reading frame. The molecular weight deduced by amino acid sequence was 242 kDa, coded with 2158 amino acid. The calculated isoelectric point was 8.9. The Vg was cut into 50 kDa and 190 kDa protein by a putative cleavage site of RXXR. Utilizing online software, SignalP,3'non-coding area is 220 bp,5'non-coding area is 37 bp. The signal peptide was consisted of 16 amino acids. The deduced primary structure indicated the content of Ser (S) up to 12.5%, the content of Asp (N) is 12.7%, the content of Glu (Q) is 8.9%. Clustal W and phylogenetic tree analysis showed that Vg in B. tabaci was most closely related with Homalodisca coagulate (86.7%), Athalia rosae (85.6%), Lethocerus deyrollei (84.5%). After we conducted the study on the gene transcription level, increased Vg transctiption gradually in B. tabaci fed on health cotton was observed.6 cDNA cloning and expression analysis of vitellogenin receptor in B. tabaciThe cDNA sequence of vitellogenin receptor (VgR) was cloned from B biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci is 5574 bp. The molecular weight deduced by amino acid sequence was about 201 kDa, coded with 1919 amino acids,5'non-coding was 201 bp. The calculated isoelectric point was 8.7. The transmembrane domain was from 1679 to 1696 amino acid at C-terminal. The VgR belong to the low density lipoprotein receptor (LDLR) family and have some conserved domains, including EGF-C, DXS/KDE, ligand-binding domain, transmembrane and the O-linked carbohydrate domain. Clustal W and phylogenetic tree analysis showed that Vg receptor in B. tabaci was most closely related with Periplaneta americana (83%), Blattella germanica (74%). The transcriptional level of VgR was detected using quantitative RT-PCR.7 cDNA cloning analysis of vitellogenin in Q and ZHJ2 biotype whitelflies B. tabaciThe cDNA sequence of vitellogenin was cloned from Q biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci is 6871 bp with 6651 bp of open reading frame. The molecular weight deduced by amino acid sequence was 246 kDa, coded by 2217 amino acid. The calculated isoelectric point was 8.9. The Vg could be cut into 50 kDa,90 kDa,150 kDa or 190 kDa proteins by two putative cleavage sites of RXXR at either 490 and/or 790 amino acid.3' non-coding area is 107 bp,5'non-coding area is 43 bp. The signal peptide was consisted of 17 amino acids. The deduced primary structure indicated the content of Ser (S) up to 15%, the Asp (N) was 12.8%, the Glu (Q) was 6.2% and the Ala (A) was 9.2%. Clustal W and phylogenetic tree analysis showed that Vg in Q biotypewas most closely related with Athalia rosae (63.4%), Lethocerus deyrollei (57.3%), Homalodisca coagulate (55.6%).The cDNA sequence of vitellogenin was cloned from ZHJ2 biotypes whitefly B. tabaci. The full length of cDNA in B. tabaci was 6721 bp with 6549 bp of open reading frame. The molecular weight deduced by amino acid sequence was 245 kDa, coded by 2183 amino acid. The calculated isoelectric point was 8.7. The Vg was cut into 50 kDa and 190 kDa protein by a putative cleavage site of RXXR. 3'non-coding area was 109 bp,5'non-coding area was 63 bp. The signal peptide was consisted of 22 amino acids. The deduced primary structure indicated the content of Ser (S) up to 12.5%, the Asp (N) was 13.5%, the Glu (Q) was 7.1% and the Ala (A) was 9.2%. Clustal W and phylogenetic tree analysis showed that Vg in ZHJ2 was most closely related with Athalia rosae (93.9%), Lethocerus deyrollei (93.5%), Homalodisca coagulate (89.9%).Comparison of cDNA sequence of three biotypes of whitefly, the invasive B and Q biotype have more serine (S) at the N-teminal from 620-640. The Q biotype has two putative cleavage sites of RXXR at either 490 and/or 790 amino acid. However, the B and ZHJ2 have only one. The Q biotype has GHN domain while the others have no similar demain. All of the three biotypes whitefly has poly S, Q and N domain at the N-terminal and C-terminal. However, the functions of these domains have not been reported clearly.8 Infection of tomato yellow leaf curl China virus affects the potential fecundity and the transcription level of Vg and VgR gene of its whitefly vector B. tabaci Gennadius (Hemiptera:Aleyrodidae)The application of light microscopy allowed us to verify that the reproductive organ of the whiteflies. The ovary of whitefly formed as soon as the adult emergence. No mature oocytes were observed in females in 1 d and the maximum number of oocytes was observed 11-14 d after emergence. According to main characteristics of oocytes at various development stages and the level of yolk content, the oocytes in ovaries were classified into four developmental types, separately named typeⅠ,Ⅱ,ⅢandⅣ. The type IV were those mature eggs. A rounded bacteriocyte sphere was transferred into oocyte at the terminal of ovariole. We compared the percentage of different type oocytes in the ovaries of two biotypes of whitefly fed on healthy tobacco with virus-infected plants after eclosion. TYLCCNV-infected plant had significant effects on the percentage various types of oocytes in the ovaries of B biotype. The markedly higher percentage of type II and III oocytes was examined in B biotype of whitefly fed on healthy host plants. In contrast, ZHJ-I biotype adult females had similar levels of status of various types of oocyte when fed on healthy and virus-infected plants. The transcription level of Vg and VgR gene was detected using semi-quantitive PCR and RT-PCR, the transcription level was higher in whiteflies fed on virus-infection plant when compare with those whiteflies fed on healthy plant.