Identification And Interaction Mechanism Of Symbiotic Related MicroRNAs In Heterorhabditidoides Chongmingensis With Its Symbiotic Bacterium Based On High-throughput Sequencing

Entomopathogenic nematodes(EPNs)are a kind of parasitic nematodes carrying symbiotic bacteria which help them to kill insect hosts.Heterorhabditidoides chongmingensis DZ0503CMFT(DZ),which is a novel EPNs member and belonging to the family Rhabditidae,has a stablely and obligately mutual relationship with its native strain DZ0503SBS1T(S1)of the species Serratia nematodiphila.Previous studies found that H.chongmingensis shows a significantly sharply declining trendency in its development,female/male ratio,reproductive rate and pathogenicity when associating with non native bacterial strain DR186(186)of S.nematodiphila,in comparing with association with its native one.MiRNAs are small,non-coding endogenous RNA molecules and reversely regulate the target gene expression at the post-transcriptional level.By now,studies have confirmed that miRNA participates in almost all of important biological processes,such as growth development,proliferation,differentiation,apoptosis and metabolism.At present,most studies of miRNA in nematode are focusing on Caenorhabditis elegans.To date,no study has been reported on symbiotic relative miRNAs between rabditid EPNs and their symbiotic bacterial strains.In this study,two small RNA libraries of monoxenic nematode-bacterium combinations,DZ-S1 and DZ-186,were respectively constructed and sequenced by Solexa sequencing technology.Identification of miRNAs and their differentially expression analysis were conducted by using bioinformatics analysis methods.There are 15744078 and 16283487 clean reads,ranging from 18 nt to 30 nt in size,in DZ-S1 and DZ-186 libraries,respectively.Totally,86 known miRNAs,representing 40 miRNA families,and 34 potential novel miRNA families were identified.In all,the expression of 30 known miRNAs and 8 potentially novel miRNAs were significantly differently in DZ-S1 and DZ-186,with most of these miRNAs being upregulated expression in the latter.The possible target genes of differentially expressed miRNAs were predicted using the software PicTar and RNAhybrid.To make clear the potential functions of the miRNA-targeted genes,gene ontology(GO)and pathway analysis(KEEN)annotations were assigned using the DAVID gene annotation tool.The terms related to development,reproduction and sex differentiation were most significantly enriched and it is consistent with our previous studies.KEGG pathway annotation results showed that MAPK signaling pathway,Wnt signaling pathway and ErbB signaling pathway are the most significantly enriched signaling pathway terms,while they are related the development,spermatogenesis,vulva development.Comprehensive the above analysis,the mechanism of symbiotic related miRNAs in heterorhabditidoides chongmingensis with its symbiotic bacterium are preliminary predicted.To validate the Solexa sequencing results,the expression patterns of 10 differentially expressed miRNAs were validated by qRT-PCR.To clear the further dynamic expression patterns of these miRNAs at different time points(6d,12d,15d,18d,24d)in nematodes,8 miRNAs were validated by qRT-PCR.The results revealed that the expression of miRNAs at different time points(6d,12d,15d,18d,24d)in DZ-S1 is stable and the change is not particularly significant while the expression of miRNAs in DZ-186 is unstable and appeared irregular changes.This study describes the novel and differential expressedmiRNAs in EPNs DZ living with two symbioticbacteria.It represents the first transcriptome-based analysis of miRNAs related to symbiosis in EPN and these findings could provide valuable information for functional characterization of miRNAs to the symbiosis.