| Wolbachia endosymbionts have formed multiple reproductive manipulation types to their hosts in the long-term evolutionary process,and one of the most important phenotypes is cytoplasmic incompatibility(CI).Sogatella furcifera and Nilaparvata lugens,the important agricultural pests,can be infected with Wolbachia w Fur with CI inducibility and Wolbachia w Lug without CI inducibility,respectively.However,limited by the inability of Wolbachia to survive in vitro independently and the differences in host background between two Wolbachia strains,their intrinsic biological property,the regulatory capacity to the same host and application potential are unknown.In this study,we stably introduced two Wolbachia strains with different reproductive manipulation capabilities into the same Ae.albopictus cell hosts.(1)Stable introduction of two Wolbachia strains respectively into cell lines.We firstly introduced two Wolbachia strains with different reproductive manipulations from rice planthoppers into Ae.albopictus cell lines,respectively。w Furinfected cell line(WFI)and w Lug-infected cell line(WLI)were successfully established.In contrast,whether these were SFCW embryos or SFC embryos,the gradual decreases of Cardinium density were observed during cell passage,which indicated that Cardinium could not be stably transinfected into cells.The stronger competitiveness of w Lug-infected cell line(WLI)than w Fur-infected cell line(WFI)during mixed culture was observed.(2)The effect of Wolbachia on the transcription of Ae.albopictusA total of 27 cell samples were sequenced by BGISEQ-500 platform,with an average of 6.66 Gb data per sample.A total of 36114 genes and 51211 novel transcripts were detected in all cell samples.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis found that up-regulated DEGs were mainly related to translation(ribosome),aging(longevity regulating pathway)and immune system(antigen processing and presentation).The stable infection and passage of Wolbachia in cells may be due to that improved cell lifespan induced by Wolbachia promoted their spread in cells.To understand the reasons for the difference in competitiveness of cell hosts infected by two different Wolbachia strains,we identified 94 up-regulated genes and141 down-regulated genes between WLI cells and WFI cells.KEGG enrichment analysis found that up-regulated DEGs were mainly related to energy metabolism,biosynthesis of other secondary metabolites,metabolism of other amino acids(e.g.,glutathione metabolism).Up-regulation of glutathione may help to delay senescence and enhance immunity to the host.Many down-regulated DEGs were related to signal transduction(e.g.,AMPK signaling pathway),which may be made it take a longer time for molecular signals from outside into cells by passing through the cell membrane,thereby promoting the rapid growth of the cells.(3)The effect of Wolbachia on the metabolism of Ae.albopictus cellSimilar numbers of up-regulated and down-regulated differential metabolites(104vs.94)were observed in the WFI/WU combination.In contrast,a low number of upregulated differential metabolites and a large number of down-regulated differential metabolites were observed in the WLI/WU combination(60 vs.283),as well as in the WLI/WFI combination(78 vs.160).This showed that WLI had different metabolic responses compared to WFI and WU.The differential metabolites between two cell lines were determined to explore the reasons for differences in cell competitiveness.KEGG enrichment analyses found that up-regulated differential metabolites were mainly related to nucleotide metabolism(purine metabolism),metabolism of cofactors and vitamins(pantothenate and Co A biosynthesis),global and overview maps(metabolic pathways),etc.;while down-regulated differential metabolites were mainly related to signaling molecules and interaction(neuroactive ligand-receptor interaction),amino acid metabolism(histidine metabolism),etc.(4)Transcriptome and metabolome data association analysisTranscriptome and metabolome data association analysis was used to explore the correlation between DEGs and differential metabolites.A total of 6 metabolites and 25 genes were significantly correlative at the set rigorous threshold,and these genes and metabolites were differential in WLI/WFI combination.Most genes related to upregulated metabolites were down-regulated in WLI cells such as alpha-mannosidase 2,prostatic acid phosphatase-like and probable cytochrome P450 9f2,which indicated that the two showed a negative correlation.Consequently,a greater number of upregulated differential metabolites and a greater number of down-regulated DEGs were present in WLI cells than in WFI cells.Low expression of these genes leading to increases of the key metabolites may be linked to an increase in cell growth and competitiveness,while the effect of high expression of genes is less clear.Combined with the above considerations,we argue that the replacement process of WFI cells replaced by WLI cells mainly included two manners when they were mixed and cultured in one shell flask(Fig.5–2).For the first case,when two cell lines were mixed and cultured,the faster growth rate of w Lug than w Fur determined the success of WFI replaced by WLI.For the second case,the infection of w Lug effectively promoted the cell growth rate of WLI,which determined the successful replacement of WFI by WLI.The faster cell division of WLI than WFI within the same period during mixed culture offered an advantage of cell number for WLI.The high cell growth rate was induced by a series of genetic and metabolite changes with the infection of w Lug.In conclusion,These results revealed the biological property of two Wolbachia strains and the potential mechanism of their inducing cell competitiveness differences,and provide a reference for understanding the interaction between Wolbachia and host,as well as the transinfection and application of Wolbachia in arbovirus vectors. |
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