| Wolbachia are endosymbiotic bacteria that infect a wide variety of invertebrates,and which can spread through populations by a process known as cytoplasmic incompatibility(CI),which results in lethality of embryos derived from crosses between Wolbachia-infected males and uninfected females or females carrying a different strains.CI is not normally induced in crosses involving females and males carrying the same strain of Wolbachia because the presence of Wolbachia can rescue embryonic lethality associated with CI.Although several models have been described to explain Wolbachia-induced embryonic lethality,the molecular mechanisms underlying embryonic lethality are not yet entirely clear Several evidence suggest that the modification of Wolbachia during spermatogenesis could be the main cause of embryonic lethality,since embryonic lethality-related defects are limited to paternal chromosome.Wolbachia is also well known for providing benefits to its host.For example,Wolbachia supply essential nutrients to their hosts,are required for successful egg development,or are essential for the production of female offspring.Other effects on arthropod hosts include protection against viruses and suppression of sterile and lethal mutants in Drosophila.Wolbachia also infect nematodes,such as in Brugia,where the symbiont is required for embryogenesis and larval development.By providing these benefits,Wolbachia enhance its own maternal transmission and has the ability to rapidly spread through a population.In the planthopper Laodelphax striatellus,an important pest of rice,Noda et al previously demonstrated that embryonic lethality levels were strong and almost complete in a laboratory setting.Among a wide range of hosts surveyed,N.lugens was found infected with Wolbachia strain wLug,whereas L.striatellus was infected with strain wStri.These closely related Wolbachia cause very similar production-related phenotypes:both Wolbachia strains can enhance host fecundity.Previous study also show that N.lugens can keep normal growth and development when they are reared at 20?-30? and the suitable temperature is 20?-27? for L.striatellus.Otherwise,the two planthopper species inhabit different areas,especially over winter.The northern borders of overwintering areas for N.lugens are around 21-25°N,but L.striatellus can overwinter in all rice-growing areas in Asia.Additionally,N.lugens can cause damage from the tropics to 42-44°N in Asia through migration.but L.striatellus cannot cause damage in tropical areas However,whether the reproductive effects of Wolbachia could also be altered under temperature stress was still not clear?To understand the molecular mechanisms of Wolbachia-induced male infertility,we selected 4-day-old male testes(when Wolbachia levels are high for analysis of transcriptome differences between Wolbachia-infected and uninfected L.striatellus.After evaluating the data,5,190 differentially expressed genes were identified as significant,including 2,266 up-regulated and 2,924 down-regulated genes.Microarray identification revealed numerous differentially expressed genes between Wolbachia-infected and uninfected samples.Among them,most candidate genes appeared to have a link to reproduction,especially comp 144942_c0(iLvE),which mediates BCAA biosynthesis;comp138099_c0(CDK1),which is a key member of cell cycle;and comp108569_c0(Lola),which can cause chromatin condensation abnormalities.Comp144942_c0(iLvE)showed the highest level of down-regulation in infected versus uninfected males.We then measured the transcription of five down-regulated genes related to reproduction in whole adult testes over time to understand their expression dynamics in individual L.striatellus,including Wolbachia-infected males and their uninfected counterparts.In uninfected L.striatellus adult testes,expression of iLvE,Lola and CDK1 showed over 0.5-fold up-regulation compared with Wolbachia-infected L.striatellus adult testes.Results indicate that these genes might participate in the process of modifying the host testes induced by Wolbachia.After knocking down iLvE by RNAi in uninfected male L.striatellus,male fertility was reduced,leading to a decrease in embryo hatching rates,but fertility was rescued in crosses between these males and Wolbachia-infected females.After knocking down other two genes by RNAi in uninfected males,male fertility was also reduced,but fertility was not rescued in crosses between these males and Wolbachia-infected females.Removal of BCAA in chemically-defined diets of uninfected males also led to a loss of male fertility.Meanwhile,low amino acid nutrition can enhance exposure time of sperm to Wolbachia in the testes to affected adult reproduction in L.striatellus by reducing the number of sperm transferred per mating by males.These results indicate that Wolbachia may decrease male fertility in L.striatellus by acting on iLvE,a key factor of BCAA biosynthesis and delay sperm maturation to modify sperm.We then investigate that whether Wolbachia can affect the female fecundity of N.lugens and L.striatellus under different temperature conditions.For both N.lugens and L.striatellus,a significant effect of female infection status was found,with infected females producing more eggs on average than uninfected females regardless of the infection status of males.Wolbachia density was stable in 3rd-5th instar ovaries of N.lugens and L.striatellus nymphs,which indicates that Wolbachia may provide stable beneficial factors to hosts.To understand the underlying mechanisms of these beneficial factors,we sequenced and compared wLug(N.lugens)and wStri(L.striatellus)genomes.Based on the Gene Ontology(GO)assignments,we found similar gene function classification between wStri and wLug.Between wStri and wLug,there were only different genes that belonged to cellular process,metabolic process,binding,and catalytic activity.To further investigate biological functions of genes in the two Wolbachia strains,sequenced genes were mapped to the Kyoto Encyclopedia of Genes and Genomes(KEGG).wStri and wLug also showed similar KEGG pathway classifications.Between wStri and wLug,there were only different genes that belonged to cellular process,metabolic process,binding,and catalytic activity.The only complete biosynthetic pathways related to nutrition synthesis in wStri and wLug were those for biotin and riboflavin synthesis,which is similar to the finding in some other Wolbachia genome wCle.Vitamin analysis also showed that Wolbachia-infected N.lugens and L.striatellus contained more riboflavin and biotin than Wolbachia-uninfected individuals,which is concordant with the genome data.We then investigate whether riboflavin and biotin participated in regulation of N.lugens and L.striatellus female fecundity under temperature stress by changing the amounts of riboflavin and biotin in artificial diets.It's consistent with our theory.In conclusion,several lines of evidence showed that Wolbachia may regulate female fecundity in L.striatellus and N.lugens under different temperature conditions by undergoing riboflavin and biotin biosynthesis in female hosts. |
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