The Role Of MicroRNAs In The Tetranychus Urticae-Wolbachia Interaction

MicroRNAs（miRNAs） are one type of small non-coding RNAs ～22 nucleotides （nt） in length which were reported in animals, plants and viruses. They regulate genes at the post-transcriptional level via base pairing to target sites within messenger RNAs （mRNAs）. Usually, the incomplete base pairing of miRNAs and target genes in animals result in repressing of mRNA translation and the complete base pairing of miRNAs and target genes in plants cause degrading of mRNA. The latest data shows that more than 30 thousands of miRNAs were reported, including 25 species of insects and three species of Chelicerata. miRNAs are involved in almost all physiological processes, including differentiation, development, metabolism, immunity, reproduction and apoptosis.Wolbachia is one kind of gram-negative endosymbionts widely distributed in arthropods which transmitted maternally. Wolbachia is known for its manipulation of host reproduction, especially cytoplasmic incompatibility （CI）. Additionally, more and more researches have found that Wolbachia can affect host fitness such as longevity, fertility and mating ability. Furthermore, some strains of Wolbachia have evolved to be essential for its host to fulfill metabolism and reproduction. The effects of Wolbachia on female host always favor the survival of the host, which in turn benefits the maintenance and spreading of Wolbachia itself.The two-spotted spider mite T. urticae is a cosmopolitan agricultural pest that feeds on more than hundreds of plant species. We previously demonstrated that Wolbachia induced strong CI and increased host fecundity in T. urticae. The former researches only explained the mechanism of CI at cytological level. However, there has been no research reported about the mechanism of Wolbachia-induced CI at the molecular level yet. Recently four articles have shown that Wolbachia manipulate the gene of Aedes aegypti through regulate host miRNAs to facilitate its own maintenance.In this study we identified differentially expressed miRNAs of T. urticae in the response of Wolbachia and between two sexes by Illumina sequencing the four small RNA （sRNA） libraries constructed with RNAs extracted from Wolbachia-infected and Wolbachia-uninfected female and male mites. By comparing the miRNA and mRNA data we were able to considerably narrow down the number of target genes of the differentially expressed miRNAs. Through analyzing the functions of the target genes, we could infer the physiological process which Wolbachia may participate in through mediating host miRNAs. The results of this study will favor our research on the mechanisms of T. urticae-Wolbachia interactions at the molecular level, including CI mechanism as well as other mechanisms that Wolbachia utilize to affect host physiological process. The results of this study are as follows:1.We constructed four sRNA libraries representing female infected mites （FI）, female uninfected mites （FU）, male infected mites （MI） and male uninfected mites （MU） by using 100% Wolbachia-infected and Wolbachia-uninfected female and male mites. Then the four sRNA libraries were sequenced through Illumina HiSeq2000 platform. By processing, more than 49 millionbase clean reads were generated from the four sRNA libraries. After sRNA annotation, we found that known miRNAs and novel miRNAs in the four libraries account for 1.55% to 23.73% and 0.66% to 10.17% of total reads of all the annotated sRNAs, respectively.2. Through bioinformatics methods,83 known miRNAs and 112 novel miRNAs were identified in the four libraries.140 miRNAs are shared by the four libraries, which including more than 48% novel miRNAs. We analyzed the miRNA expression level in four comparisons （FI versus （vs） FU, MI vs MU, MI vs FI and MU vs FU）. In total,91 miRNAs showed significant change in expression level between FI and FU, with 59 miRNAs down-regulated and 32 miRNAs up-regulated. However, only 20 miRNAs expressed differently in MI and MU, with 9 miRNAs down-regulated and 11 miRNAs up-regulated. 13 differential miRNAs were both found in female and male comparisons. Besides,70 miRNAs showed significant expression level between female and male mites.3. After prediction target genes for all the differential miRNAs, we analyzed these data with the trascriptomes data obtained from the same RNA samples; this is called miRNA-mRNA combined analysis. By intersecting target genes data of differential miRNAs and differential mRNAs data from the trascriptomes, we thoroughly and deeply predicted the potential target genes of the differential miRNAs. As a result,90 target genes were found to be potentially negatively regulated by differential miRNAs in the comparison FI vs FU,9 target genes were found to be potentially negatively regulated by differential miRNAs in the comparison MI vs MU. Further GO and KEGG analysis of these target genes revealed the potential functions of these genes. GO results signify these genes are mainly related to binding, catalytic activity, and metabolic and cellular processes. KEGG pathways enriched genes involved in degradation of valine, leucine and isoleucine, phenylalanine metabolism, sulfur metabolism, lysosome function and so on. KEGG analysis concluded that the only shared potentially negatively-regulated target gene in both female and male comparisons is tetur09g06680, which may be regulated by novel₁6.4. The expression level of the 14 selected miRNAs and one target gene tetur09g06680 were checked by qRT-PCR （reverse transcription quantitative polymerase chain reaction）. The results showed that novel₁26, novel₃2, novel₃6 and novel₈9 were greatly decreased （P<0.001） in infected lines, novel₁6, novel₈9, novel₁09 and novel₄3 changed more dramatically in infected males than in infected females while novel₁26, novel₃2 and novel₃6 decreased more dramatically in infected females than in infected males. Three （novel₁01, novel₈0 and novel₂1） of the five selected miRNAs exclusively differentially expressed between FI and FU were down-regulated in infected female mites. According to our qRT-PCR results, terur09g06680 was found to be up-regulated in both infected female and male lines, which was consistent with our sequencing data. This indirectly indicates that novel₁6 potentially represses target gene tetur09g06680. Taken together, those results confirmed that Illumina technology accurately reflected the abundance of the miRNAs and mRNAs.