Combined Effects Of Continue High Temperature Stress And Endosymbiotic Wolbachia Infection On Drosophila Melanogaster

With the warming of global temperature,the relationship between temperature and ecological environment has attracted more and more attention from scholars.Temperature is not only an essential factor in the natural environment,but also plays a fundamental role in the dynamics of host-pathogen interactions.Wolbachia endophytes are widely distributed in arthropods.Recently,there are lots of reports about the influence on host reproduction and fitness of Wolbachia.Although some studies have reported that temperature affects the distribution on host of Wolbachia,there is little research on the interaction between high temperature and Wolbachia.In order to explore the interaction effects of high temperature stress and Wolbachia infection on the host,this paper takes Drosophila melanogaster as the research object,according to different temperature conditions(room temperature:25°C,high temperature:31°C)and Wolbachia infection status(Wolbachia infection:W⁺and Wolbachia uninfected:W^-),4 treatment combinations are set:room temperature uninfected(25?W^-),room temperature infection(25?W⁺),high temperature uninfected(31?W⁺),high temperature infection(31?W⁺).The biological characteristics of Drosophila melanogaster(reproduction,growth and development,body weight and body length of adults and adult survival)differences on three consecutive generations of different treatment combinations were studied.By using molecular to study the differences in the density of Wolbachia in Drosophila on high temperature stress for 3 consecutive generations.Through the study of the effects of the interaction of high temperature and Wolbachia infection on Drosophila melanogaster,it is possible not only to explore the effects of the interaction between the two on the reproduction and reproduction of D.melanogaster,but also enrich the research on the sensitivity of high temperature to Wolbachia,but also for more Make good use of Wolbachia to provide a theoretical basis for comprehensive prevention and control of agricultural and forestry pests.The main content and results of this research are as follows:1.Effect of Wolbachia on the biological characteristics of Drosophila at room temperature.The tested and uninfected fruit flies of Wolbachia were placed in an artificial climate incubator(25±0.5?,RH:40±5%,L:D=14:10),the oviposition amount,hatching rate,developmental durations,pupation rate and emergence rate,as well as the lifespan and survival rate,weight and length of the adult.The results showed that:Wolbachia infection significantly reduced the oviposition amount,hatching rate,pupation rate and emergence rate of flies.The developmental durations of flies from hatching to emergence also increased significantly.Wolbachia infection significantly reduced the lifespan of fruit flies and the survival rate of flies.Wolbachia infection had no effect on the body weight and length of female flies,but significantly reduced the body weight of male flies and body length.2.Interaction between continuous high temperature stress and Wolbachia infection on Drosophila.Combine the 4 treatments(25?W^-?25?W⁺?31?W⁺?31?W⁺)in an incubator(RH:40±5%,L:D=14:10)for 3 consecutive generations,separately count oviposition amount,hatching rate,developmental durations,pupation rate,emergence rate,survival rate,weight and length of the adult in each generation.The results showed that:high temperature increased the oviposition amount of F₁^-and F₁⁺flies,but reduced the hatching rate,pupation rate,emergence rate,shortening developmental durations.Continuous high temperature stress to the F₂and F₃generations,the oviposition amount,hatching rate,pupation rate,emergence rate of flies were significantly reduced,and the developmental durations was significantly prolonged.Wolbachia infection not only significantly reduced oviposition amount,hatching rate,pupation rate and emergence of flies at room temperature,it also significantly reduced this at high temperatures.The two-way analysis of variance showed that high temperature and Wolbachia infection had an interactive effect on the hatching rate,development period and emergence rate of F₁,F₂,F₃oviposition amount,hatching rate,developmental durations,pupation rate,emergence rate all have an interactive effect.Both high temperature stress and Wolbachia infection significantly reduced the lifespan of flies.Wolbachia infection and high temperature stress also significantly reduced F₁,F₂and F₃survival rate of flies.Two-way analysis of variance high temperature stress and Wolbachia infection have no interactive effect on the lifespan and survival rate of F₁generation flies,but there is an interactive effect on the lifespan and survival rate of F₂and F₃generation flies effect.High temperature significantly reduced weight and body length of female flies.Wolbachia infection has no significant effect on weight and body length of female flies at room temperature,but Wolbachia infection significantly reduces weight and body length of female flies at high temperature.Two-way analysis of variance showed that high temperature stress and Wolbachia infection had an effect on the body weight of F₁,F₂,and F₃female flies and body length have an interactive effect.High temperature significantly reduced weight and body length of male flies.Wolbachia infection significantly reduced weight and length of male flies at room temperature or high temperature.Two-way analysis of variance showed that high temperature stress and Wolbachia infection had an effect on weight of F₁,F₂,and F₃male flies and body length have an interactive effect.3.Effect of continuous high temperature stress on Wolbachia infection rate and density.The newly emerged adult fruit flies were randomly selected from the 31?W⁺,and32 flies were selected in each generation,then DNA was extracted to detect the effect of high temperature stress on Wolbachia infection rate.RNA was extracted and reverse transcribed to c DNA for quantitative PCR to detect density of Wolbachia in each generation of flies.Each generation performed 3 biological replicates,and each biological replicate performed 3technical replicates.The results showed that:three consecutive generations of high temperature stress reduced Wolbachia infection rate of Drosophila from 100%to 78.125%,indicating that high temperature stress can effectively reduce Wolbachia infection rate of Drosophila.According to the CT values detected by RT-q PCR of in F₁⁺?F₂⁺and F₃⁺flies,the density of Wolbachia in different generations were calculated by the method of 2^-??CT,W⁺:3.142±0.077,F₁⁺:1.981±0.040,F₂⁺:1.001±0.001,F₃⁺:0.165±0.042.The independent sample t-test was used to analyze the difference of Wolbachia density in each generation of Drosophila.The results show that continuous high temperature stress can significantly reduce the density of Wolbachia in F₁⁺?F₂⁺and F₃⁺flies.