Mechanisms Of Bemisia Tabaci-Symbionts-CCYV Interactions

There are a large number of microorganisms inhabiting insects,and these microorganisms and host insects have formed interdependent symbiosis in the process of long-term coevolution.Symbiotic bacteria participate in almost all life activities of the host insect,not only can regulate the host’s nutrition,metabolism,reproduction,etc.,but also play important roles in the host’s adaptation,evolution and diversity.Symbionts can also directly or indirectly affects the virus transmission by regulating the host’s immune or physiological functions.Bemisia tabaci is a super invasive pest of many fruits and vegetables and one of the most important vectors of plant viruses,causing serious crop losses in agricultural production.Among the plant viruses,cucurbit chlorotic yellows virus(CCYV)is specifically transmitted by B.tabaci in a semi-persistent mode.In recent years,CCYV has caused serious damage to melon crops in many countries and regions in the world.However,it is not clear whether the virus can regulate B.tabaci symbiotic bacteria related to virus transmission.This research adopts techniques and methods of insect biology,insect physiology,molecular biology,chemical ecology,and macro-genomics,by using B.tabaci Q type with or without removing the symbionts,CCYV and cucumber as the research system,effects of CCYV on symbiont diversity in B.tabaci and its honeydew and mechanism of whitefly-CCYV-plant interactions mediated by secondary symbiont Rickettsia were investigated.The results can be concluded as follows:(1)Effects of CCYV on composition and diversity of symbiotic bacteria in B.tabaci.Metagenomic sequencing was performed on B.tabaci carrying CCYV and healthy B.tabaci to intuitively compare the difference of endosymbiotic bacteria before and after B.tabaci was infected.After B.tabaci was infected with CCYV,the relative abundance of proteobacteria and actinomycetes increased significantly,while the relative abundance of Bacteroidetes decreased significantly.At the genus level,the relative abundance of Rickettsia in B.tabaci increased significantly after CCYV infection,while the relative abundance of Hamiltonella and Acinetobacter decreased significantly.At the same time,CCYV infection significantly reduced the richness of the symbiotic bacteria community,confirming that CCYV significantly affected the composition,structure and species diversity of the symbiotic bacteria community in B.tabaci.(2)Effects of CCYV on microbial composition and diversity in honeydew of B.tabaciHoneydew secreted by CCYV-carrying B.tabaci and healthy B.tabaci was collected,and the bacteria in honeydew were sequenced by metagenomic sequencing.It was detected that some microorganisms in B.tabaci honeydew were related to endosymbionts,and there were many other bacteria.After B.tabaci were infected with CCYV,the relative abundance of Proteobacteria in secreted honeydew was significantly increased,while the relative abundance of Actinobacteria and Firmicutes was significantly decreased.At the genus level,after CCYV infection,the relative abundances of Brachybacterium,Paraburkholderia and Herbaspirillum in honeydew decreased significantly,while the relative abundance of Pantoea increased significantly,becoming the dominant genus.Meanwhile,infection with CCYV had no significant effect on the richness of honeydew microbial flora of B.tabaci,but significantly reduced its diversity,confirming that CCYV significantly affected the composition,structure and species diversity of honeydew microbial flora secreted by B.tabaci.(3)Effects of Rickettsia on B.tabaci-plant-virus interactions.First,the biological characteristics(including egg production,egg hatching rate,and survival rate)of R+(with Rickettsia)and R-type(removing Rickettsia)B.tabaci on infected and healthy plants were observed,recorded and analyzed.The number of eggs laid by the R-type B.tabaci was significantly lower than that of the R+ type B.tabaci,and the R-type B.tabaci had the lowest egg laying rate on healthy cucumber plants.The egg hatching rate and survival rates of the R+ and R-type B.tabaci were not significantly different.Secondly,in the analysis of R+ and R-type B.tabaci’s free selection tendency to infected and healthy cucumber plants,it was found that the B.tabaci carrying Rickettsia tended to feed on the infected cucumber plants significantly.However,B.tabaci without Rickettsia showed no obvious selection tendency to susceptible and healthy hosts.Furthermore,the amount of honeydew secreted by R+ type and R-type B.tabaci after feeding on the susceptible plants and healthy cucumber plants was measured,and it was found that the amount of honeydew secreted by the R+ type and R-type B.tabaci after feeding on the susceptible plants was significantly higher than that of the healthy plants,and whether the R-type B.tabaci fed on the susceptible plants or healthy plants,The amount of honeydew secreted by B.tabaci was significantly lower than that of R+ type.Finally,EPG system was used to monitor the feeding behavior of R+ and R-B.tabaci on infected and healthy cucumber plants.It was found that the total duration of NP and the total number of PD were significantly different between the two kinds of B.tabaci and healthy cucumber plants.These results jointly confirmed that CCYV not only enhanced host plant attraction to B.tabaci,but also significantly increased feed intake of B.tabaci,and also proved that Rickettsia could affect host selection preference and feeding behavior of B.tabaci.(4)The effect of Rickettsia on the CCYV transmission ability of B.tabaci.The established real-time RT-PCR method was used to detect the virus-carrying status of a single B.tabaci and its host plant,and it was applied to determine the regulatory ability of the symbiotic bacteria Rickettsia on the transmission of CCYV by B.tabaci(including the time of acquisition,the time of holding the virus,the time of transmission,and the poisoning efficiency).First,the difference in the acquisition rate and amount of B.tabaci(R+ type)without eliminating the symbiotic bacteria Rickettsia was analyzed,and the results showed that: There was no significant difference between R+ type and R-type on CCYV acquisition rate(P > 0.05),but the virus titer of R-type in B.tabaci was significantly decreased after feeding on susceptible cucumber plants for 24 h and 48 h(P < 0.05).Next,the persistence time of R+ type and R-type B.tabaci to CCYV was determined.The results showed that after eliminating the commensal bacteria Rickettsia,there was no significant difference in the retention time of the virus(P > 0.05).Both B.tabaci populations could last for 15 days,but the virus titer of R-type tabaci were significantly decreased after feeding on cotton plants for 9 days,12 days and 15 days(P < 0.05).Finally,the differences in the transmission rate of R+ type and R-type B.tabaci were compared,and the transmission efficiency of B.tabaci after eliminating Rickettsia bacteria decreased from77.78% to 53.33%.These results indicated that the secondary symbiotic bacteria Rickettsia could significantly improve the ability of B.tabaci to transmit CCYV,increase the amount of virus acquired by B.tabaci,and slow down the rate of virus titer decrease,but had no significant effect on the ability of B.tabaci to acquire virus and the duration of virus retention.In summary,the research in this paper proves that CCYV significantly affects the composition and diversity of symbiotic bacteria in B.tabaci.Rickettsia has obvious regulatory effects on the reproductive function,host preference,feeding behavior and feeding amount of B.tabaci,and significantly improves the ability of B.tabaci to transmit CCYV.