| Objective:Wolbachia is a gram-negative endosymbiotic bacterium that is widely distributed in arthropods and nematodes and can be maternally transmitted.Wolbachia can regulate host reproduction in many ways,and the most prominent one is the induced cytoplasmic incompatibility(CI)in its host.In the CI phenomenon,Wolbachia cause varying degrees of embryonic mortality,resulting in reduced numbers of offspring.Due to this unique way of reducing the reproduction of vector insects,Wolbachia has been used on a large scale for the biological control of vector-borne infectious diseases such as Dengue fever and Zika in recent years.For effective biological control the induction of high levels of CI is required.Wolbachia-induced CI levels in its host are affected by a variety of factors,including Wolbachia strains,host genetic background,and host growth stage.In this study,the D.melanogaster Canton-S strain and the Wolbachia wMelCS strain were used to determine the Wolbachia density in D.melanogaster with different growth stages,and to investigate the relationship between D.melanogaster age and CI occurrence.Our study may help establish an effective CI induction model.Methods:Part One.Wolbachia density and distribution in reproductive tissues and whole body of Drosophila melanogaster with different ages1.Determination of Wolbachia density in reproductive tissues and whole body of D.melanogaster with different ages by competitive PCRFirstly,based on the DNA sequence of D.melanogaster actin gene and Wolbachia outer membrane protein wsp gene,three pairs of primers are designed for each gene and used in PCR to generate the competitor which is 20%shorter than original sequence in length.The genomic DNA of D.melanogaster and its reproductive tissues at days 1,5,10,15 and 20 were extracted and subjected to competitive quantitative PCR.Gray-scale analysis of PCR bands was performed using Quantity One,and statistical analysis was performed using GraphPad Prism.2.Western Blot analysis of the expression level of Wolbachia outer membrane protein Wsp in reproductive tissues of D.melanogaster with different ages2.1 Construction of prokaryotic expression system to express Wolbachia outer membrane protein WspDNA fragment encoding Wolbachia outer membrane protein Wsp were amplified by PCR with D.,melanogaster genomic DNA as template and the specific primers incorporated with cleavage site(EcoR I and Not I).DNA fragments were cloned into prokaryotic expression vectors pET28a(+).The recombinant plasmid was sequenced and transformed into E.coli BL21(DE3)competent cells for expression.2.2 Expression and purification of Wolbachia outer membrane protein Wsp E.coli BL21(DE3)culture with recombinant plasmid was induced by IPTG at 37? to express protein.Wsp were purified by Nickel affinity chromatography followed by SDS-PAGE.2.3 Preparation of rabbit polyclonal antibodyRabbit polyclonal antibody against Wolbachia Wsp was made commercially by using purified recombinant Wsp as antigen.The specificity of antibody was determined by Western Blot analysis2.4 Collection of Drosophila melanogaster reproductive tissues and extraction of proteinD.melanogaster reproductive tissues were collected for 1 d,5 d,10 d,15 d and 20 d.Their reproductive tissue proteins were extracted and detected by Western Blot using mouse antibody against D.melanogaster tubulin and rabbit antibody against Wolbachia Wsp.3.Distribution of Wolbachia in Drosophila melanogaster reproductive tissuesFluorescence in situ hybridization was used to observe the location and distribution of Wolbachia during oocyte maturation in the ovarian tubules and testis of D.melanogaster.Part Two.The relationship between the degree of Wolbachia-induced cytoplasmic incompatibility and the age of Drosophila melanogasterMale D.melanogaster with the age of 1 and 15 days mate with 2-3 day old female D.melanogaster for 24 hours.Embryonic hatching rates were calculated.Results:?.Wolbachia density and distribution in reproductive tissues and whole body of Drosophila melanogaster with different ages1.Determination of Wolbachia density in reproductive tissues and whole body of D.melanogaster with different ages by competitive PCRThrough three rounds of PCR reaction,the competitors for D.melanogaster actin reference gene and Wolbachia outer membrane protein wsp gene were successfully prepared.Competitive PCR results showed that the density of wMelCS was positively correlated with D.melanogaste age in female individuals,male individuals and testis.However,this correlation was not observed in ovary.Western Blot analysis of the expression level of Wolbachia outer membrane protein Wsp in reproductive tissues of D.melanogaster with different agesWestern Blot analysis showed that the expression of wMelCS Wsp in the ovary of female D.melanogaster did not change significantly with D.melanogaste age,but the expression of wMelCS Wsp in male Drosophila testis increased with age,which was consistent with the PCR results.3.Distribution of Wolbachia in Drosophila,melanogaster reproductive tissues wMelCS was evenly distributed in the 1st and 2nd regions and located at oocyte in 3rd region of germarium of ovariole.wMelCS was positioned at the posterior pole at spherical phase during oogenesis.With further development to the goblet phase,hemispherical phase wMelCS was present throughout the oocyte.wMelCS was distributed throughout the oocyte cell during the yolk cell degeneration phase and has the highest density at the posterior pole.wMelCS was mainly distributed in the spermatozoa germinal center and the front of the testicular duct in 1 d Drosophila testis;wMelCS in the 15 d Drosophila testis mainly located in the posterior part of the testicular duct.Wolbachia does not exist in the vas deferens.?:The relationship between Wolbachia-induced cytoplasmic incompatibility and the age of Drosophila melanogasterThe results showed that as male flies got older,the degree of cytoplasmic incompatibility decreased.Conclusions:1.Quantitative analysis were used for the first time to systematically study the changes of wMelCS density in Drosophila individuals and their reproductive tissues with different ages.It was found that the density of wMelCS in female Drosophila individuals,male Drosophila individuals,and testis increased with age.However,in female Drosophila ovaries,the density of wMelCS did not change significantly with age.2.The study on the distribution of wMelCS at each stage of the oogenesis of Drosophila not only further proves that Wolbachia can enter the oocyte from the vegetative cells,but also shows that the Wolbachia enters the oocyte from the vegetative cell mainly in the hemispheric and yolk cell degeneration phase of oogenesis.3.The first study of the distribution of wMelCS in the testis of different ages showed that wMelCS was mainly distributed in the spermatozoa germinal center and the front of the testicular duct in 1 d Drosophila testis;wMelCS in the 15 d Drosophila testis mainly located in the posterior part of the testicular duct,In addition,we found that the degree of CI caused by wMelCS weakened as the fruit fly ages.These work provide an important basis for establishing a high-level CI model. |
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