2.1 Culture condition
The susceptive strain of S. avenae used was from the greenhouse for more 10 years, and has never been exposed to any insecticides. The culture condition was mentioned as previously.23
2.2 Imidacloprid treatments
Leaf-dipping with aphids method was conducted with minor modifications as previously described Chen et al.24 Leaf-dipping with 3rd instar nymphs aphids were immersed in the imidacloprid solution (0.5 mg L-1) for 5-10 s. And their imidacloprid-treated counterparts treated with distilled water as the control were also collected. Surviving aphids (N = 6 for each sample), including imidacloprid challenge and counterparts were collected at the same time points (24 h).
2.3 RNA isolation, miRNA library construction and Illumina sequencing
Total RNA was extracted from controls and imidacloprid treatments of S. avenae using TRIzol reagent (Invitrogen, Shanghai, China) following the manufacturer’s protocol and re-suspended in nuclease-free water 1% agarose gels were monitored to determine if RNA extracted was degradated and contaminated, and then purity, concentration and integrity of RNA were measured by NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE), Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA) and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. Approximately 3 μg of total RNA as input material of each sample was used for constructing the small RNA library and conducted in Novogene, Beijing, China as previous description25.
2.4 Bioinformatics analysis, miRNA prediction, and miRNA target prediction.
S. avenae miRNAs, were confirmed by miRDeep2 software.26 Raw reads from the 4 libraries, including imidacloprid treatments and controls (2 biological replicates of each sample), were sent as insert into miRDeep2, and then the data (each library) was further separately analyzed. The default options and settings were used to performed the miRDeep2 analysis. And the sequences with polyA tails of the raw reads, the length of miRNAs ranging from 18 to 30 nt was selected for further being analyzed after the adaptor sequences was trimmed, and removed snRNA, rRNA, snoRNA and tRNA. The sequences, mapping to the mature miRNAs in the premiRNAs and miRBase, and mapping to the Acyrthosiphon pisum genome would be identified to be as known mature miRNAs. The clean reads identified of S. avenae transcriptome has been sent to NCBI/SRA database, the accession number was SRP182781. Miranda performed the target genes prediction of miRNA.27 The putative mRNAs that targeted by miRNAs were predicted against S. avenae transcriptome nucleotide sequences. 2 software programs were conducted for miRNA target prediction, miRanda28 and RNAhybrid.29 The predicted miRNA target genes would be selected for further analysis. And then the target genes predicted would be aligned by the BLASTX program of NCBI, as well as mapping and annotation of the gene sequences were conducted using BLAST2GO.30 The Gene Ontology (GO) database, Genomes (KEGG) pathway and Kyoto Encyclopedia of Genes were used to further analyse putative identified target genes of miRNAs.
2.5 Differential expressed miRNAs between controls and imidacloprid treatments of S. avenae
In order to obtain the miRNAs of aphids affected by imidacloprid, the expression level of miRNAs was performed between controls and treatments of S. avenae. The edgeR software (3.10.2) obtained from Bioconductor version 3.1 was used to analyze the read counts of identified miRNA.31
The P-values was calculated by the Benjamini-Hochberg method.32 P-value corrected was set at 0.05 as the threshold for expressed difference by default significantly. Normalization of miRNA counts between libraries of controls and treatments was executed according to the total number of reads across libraries in silico. Normalized expression = Actual miRNA count/Total count of clean reads×106. A false discovery rate (FDR) of the miRNAs <0.05, and a fold change ≥2 was deemed to be significant.
2.6 Quantitative real-time PCR (qPCR) validation
To validate the miRNA data obtained by the deep sequencing, 3 miRNAs were selected to confirm their expression by qPCR. Total RNA, first strand cDNA synthesized, SYBR Green Master Mix (miScript SYBR Green PCR Kit, Qiagen) and qPCR procedure determined was described as previously.33 Relative expression quantification was calculated by the 2−∆∆Ct method.34 U6 snRNA was used to normalize expression quantiy of miRNAs as an endogenous control. The primers designed were showed in Table 1.
2.7. MiRNA inhibitor feeding in vitro, and subsequent impacts on imidacloprid susceptibility
The rearing method and the artificial diet used were as previously described.35 with minor modifications. Both ends of transparent glass tube were open, sterilized and used as the feeding device (4 cm in length, 2.5 cm diameter). 25% sucrose as artificial diet was fit into 2 layers of parafilm sealed in the one end of the feeding device, and then healthy apterous adults were transfered to the device for rearing covered by the mesh to prevent them from escaping. The aphids were reared under the condition described previously. Each sample contained three replications.
To evaluate the inhibitory effect of miRNAs after the feeding of miRNA inhibitors, the artificial diet with miRNA inhibitors at a 2.5 mM/L final concentration, 50 healthy apterous aphids were placed to the artificial diet mixed with miRNA inhibitor, and the NC-inhibitor (a negative control) were used as the control; Following the feeding of 24 h, the aphids survival were collected for RNA extraction, 3 replicates were carried out.
To evaluate the affects of miRNAs modulated on sensitivity of S. avenae to imidacloprid, 50 healthy apterous adults were placed to the artificial diet that contained imidacloprid (0.1 mg/L) with a 2.5 mM/L final concentration miRNA inhibitor, the NC-inhibitor (a negative control) were used for the control. 3 replicates were carried out, and mortality was caculated at 48 h. The miRNA inhibitors used were provided by Sangon Biotech Co., Ltd (Shanghai, China).