Productive Manipulation And Interaction Of Endosymbiont Wolbachia And Cardinium In Bemisia Tabaci

The whitefly Bemisia tabaci（Gennadius）（Hemiptera:Aleyrodidae）is a cryptic species complex,including members that are global importance pests due to its cause destructive damage,like directly feeding,honey dew secreted induced plant mildew stains,and transmission of plant viruses.In this study,the aboriginal B.tabaci have been used as research objects,focusing on the the reproductive regulatory function of endosymbiont Wolbachia,we studied that the reproductive regulatory function and Interaction relationship of endosymbiont Wolbachia and Cardinium in the Asia II 7 B.tabaci,and the reproductive regulatory function of endosymbiont Wolbachia in the Asia II 1 B.tabaci by using the PCR,quantitative PCR and fluorescence in situ hybridization（FISH）technique,combined with the method of ecological experiment research.The dynamic changes of the concentrations and positions of endosymbiont Wolbachia and Rickettsia were studied in different time eggs of Asia II 1 B.tabaci.The main results were as follows:1.Phylogenetic analysis and multilocus sequence typing of endosymbiont Wolbachia in the four indigenous whiteflyUsing PCR technique to detect infection statu of endosymbiont Wolbachia in the AsiaⅠ,AsiaⅡ1,AsiaⅡ7 and China 2 aboriginal whiteflies,the results showed that the four whitefly have infected the endosymbiont Wolbachia.The Wolbachia sequence type of the AsiaⅡ1,AsiaⅡ7 and China 2 whitefly were 378,and Wolbachia sequence type of AsiaⅡ1 was 377.the phylogenetic analysis of wsp gene and MLST polygene were indicated that Wolbachia of infected four aboriginal whiteflies belonged to the group B,and Wolbachia was predicted to be able to induce CI.2.The detection of kinds of endosymbionts in the AsiaⅡ1 and AsiaⅡ7 B.tabaciUsing PCR to detection species of endosymbionts infection in the AsiaⅡ1 and AsiaⅡ7 B.tabaci shows that AsiaⅡ7 B.tabaci was infected with one primary endosymbiont（Portiera）and four secondary endosymbionts（Cardinium、Wolbachia、Hemipteriphilus and Fritschea）,and infection rate of Cardinium,Hemipteriphilus and Wolbachia were 100%.Besides infection the primary endosymbiont（Portiera）,the AsiaⅡ1 B.tabaci was infected with the our secondary endosymbionts（Rickettsia、Wolbachia、Hemipteriphilus and Fritschea）,and The infection rates of Rickettsia and Hemipteriphilus were 100%,while the average infection rates of Wolbachia and Fritschea were 68.3%and 38.3%respectively,and the female infection rate was higher than the male.3.Screen and setup of different endosymbiont infection line in the AsiaⅡ7 B.tabaciUsing the PCR technology,two sub-lines that coinfected with Cardinium and Wolbachia(I_CWH and I_CWL)were established in laboratory by“single-pair screen”method,an uninfected laboratory population（I_U）and a Wolbachia infection lines（I_W）were established by rifampicin treatment and“single-pair screen”method.The detection by q PCR and FISH technology revealed that Wolbachia was detected in both male and female of I_CWHand I_W infection lines,Cardinium was detected only in male and female of I_CWH infection line,Wolbachia and Cardinium weren’t detected in male and female of I_U infection line.The titers of Wolbachia in male adults were higher than female adults,but the titers of Cardinium in the female adults were higher than male adults.the Cardinium and Wolbachia endosymbionts were simultaneously localized in the bacteriocytes that placed in the abdomen of adults and nymphs of Asia II7 B.tabaci I_CWH line,While in the I_W line of Asia II7 B.tabaci,only Wolbachia was visualized,which localized in the bacteriocytes with a confined pattern in the abdomen of adults and nymphs.A complete（100%）maternal transmission of Cardinium and Wolbachia was observed in both the I_CWH and I_W lines.4.Effects of different endosymbionts infectionon biological parameters of Asia II7 B.tabaciThe arrhenotoky experiment of Asia II7 I_U,I_W and I_CWH virgin females revealed that the averaged fecundity,number of embryonic mortality and male progeny were decreased,as the species of endosymbiont infection of Asia II7 B.tabaci were increased,while the nymphal mortality was higher in I_CWH line than that of I_U and I_W lines.The intra-line amphigenesis experiment of Asia II7 I_U,I_W and I_CWH virgin females revealed that the averaged fecundity,number of embryonic mortality and nymphal mortality were increased as the species of endosymbiont infection of Asia II7 B.tabaci were increased,but the female progeny and female sex ratio in I_CWH amphigenesis were significantly higher than that of I_U,I_Wlines.5.Endosymbiont Cardinium induced male killing.Quantitative PCR results showed that the titers of Cardinium in the I_CWH adults,regardless the gender,were significantly higher than that of I_CWLadults,but for the titers of Wolbachia,no significant differences was recorded between the female adults of I_CWH and I_CWL lines,same between the male adults of I_CWH and I_CWL lines.the number of nymphal mortality of the I_CWH progeny was obviously higher than that of I_CWL progeny in arrhenotoky experiment.Comparing to the amphigenesis of virgin I_CWL,the amphigenesis of virgin I_CWH produced significantly less males and more females,has higher number of nymphal mortality and distinctly higher female sex ratio,ndicating again that the higher Cardinium infection led to lower male sex ratio due to its male-killing function.6.Endosymbiont Wolbachia induced cytoplasmic incompatibility（CI）.The copulation experiment of I_U×I_W and I_U×I_CWH virgin males and females results revealed that the incompatible cross♀I_U×♂I_W reproduced the fewest progeny,has the highest embryonic mortality,the highest nymphal mortality and the lowest female sex ratio when compared with the contral（♀I_U×♂I_U）.The incompatible cross♀I_U×♂I_CWH resulted in highed nymphal mortality and lowest female sex ratio than the contral（♀I_U×♂I_U）.All the results indicated that Wolbachia endosymbiont in the I_W and I_CWH lines of Asia II7 B.tabaci has the function of cytoplasmic incompatibility and respective the levels of CI were 63.55%and 11.42%7.Effects of Cardinium coinfection on the CI strength of WolbachiaThe incopulation experiment of I_W and I_CWH line results revealed that the♀I_U×♂I_CWHcopulation has significantly higher oviposition,much more male and female progeny,and has significantly lower embryonic mortality and nymphal mortality,the CI strength and CI induced post embryonic mortality in♀I_U×♂I_CWH line were markedly lower than that of♀I_U×♂I_Wline.All the results clearly indicated that,the Cardinium induced male-killing function reduced the level the Wolbachia induced CI in the I_CWH line.The outcomes of the Cardinium-induced male killing and Wolbachia-induced CI resulted in increasing the nymahal mortality.Cardinium-induced male killing resulted in more female offspring while Wolbachia induced CI resulted in less female offspring,so the antagonistic interaction of these two endosymbionts in the reproductive manipulation of whitefly host.8.Endosymbiont Wolbachia induced cytoplasmic incompatibility in the AsiaⅡ1 B.tabaciA Rickettsia infection lines（I_R）and A coinfected with Wolbachia and Wolbachia(I_WR)were established in laboratory by“single-pair screen”method.The detection by q PCR technology revealed that the titers of Wolbachia in the I_WR adults,regardless the gender,were significantly higher than that of I_Radults.but for the titers of Rickettsia,no significant differences was recorded between the adults of I_WR and I_R lines.The copulation experiment of I_WR×I_R virgin males and females results revealed that the incompatible cross♀I_R×♂I_WRreproduced the fewest progeny,has the highest embryonic mortality,the highest nymphal mortality and the lowest female sex ratio when compared with the contral（♀I_R×♂I_R）.All the results indicated that Wolbachia endosymbiont in the I_WR line of Asia II7 B.tabaci has the function of cytoplasmic incompatibility and the levels of CI were 48.72%.9.the dynamic charge of titers and location of endosymbiont Wolbachia and Rickettsia in the different times eggs of Asia II 1 B.tabaciThe dynamic charge of titers and location of Wolbachia and Rickettsia were detected by q PCR and FISH,which results showed that the titers of Wolbachia and Rickettsia in the3-120 h eggs changed presented a"w"type,the titers of both the primary endosymbiont（Portiera）and the secondary endosymbiont（Wolbachia and Rickettsia）have a"descending–ascending-descending-ascending"change process,among which the titers of the endosymbiont（Portiera,Wolbachia and Rickettsia）were the highest in the 3 h eggs and the lowest in the 9 h eggs.The location of Wolbachia and Rickettsia in the egg was the change from eggstalk to egg basal,and then from the egg basal to egg end,and finally back to the middle of egg.