2.1 Culture conditions
The susceptible strain of S. avenae used was maintained in a greenhouse for more than 10 years and has never been exposed to any insecticides. The culture conditions used were described previously.23
2.2 Imidacloprid treatments
To test the susceptibility of S. avenae to imidacloprid, the leaf-dipping method was conducted as previously described by Chen et al.24 with minor modifications. Imidacloprid was dissolved in acetone, and then serially diluted with 0.05% (v/v) Triton X-100 in water. Wheat leaves were cut into pieces 20 mm long. The leaves with aphids were dipped into 0.05% (v/v) Triton X-100 in water with 0.5 mg/L imidacloprid (LC10 concentration, Table S1) or without imidacloprid as a control. The leaves with treated aphids were put into a glass tube (6 cm in length, 2 cm diameter) and the open end was covered with cotton to prevent insect escape. Surviving third instar nymphs (N = 5 for each treatment) were collected at 24 h after imidacloprid or mock challenge. Aphids were flash frozen in liquid nitrogen before being stored at -80℃ for RNA extraction. The experiment was replicated three times.
2.3 RNA isolation, miRNA library construction and Illumina sequencing
Total RNA was extracted from control and imidacloprid-treated S. avenae using TRIzol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol and resuspended in nuclease-free water. Electrophoresis on 1% agarose gels was used to determine if the extracted RNA was degraded or contaminated, and then purity, concentration and integrity of the RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE), a Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), and a RNA Nano 6000 Assay Kit with the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. Approximately 3 μg of total RNA from each sample was used as input material for constructing the small RNA library, and the platform used for sequencing was conducted in Novogene, Beijing, China as previously described.25
2.4 Bioinformatics analysis, miRNA prediction, and miRNA target prediction.
S. avenae miRNAs, were confirmed using miRDeep2 software.26 Raw reads from the four libraries (imidacloprid treatment and control, two biological replicates each) were used as input for miRDeep2, and the data from each library were separately analyzed. The default options and settings were used to perform the miRDeep2 analysis. The raw read sequences with polyA tails and miRNAs with lengths ranging from 18 to 30 nt were selected for further analysis after trimming adaptor sequences and removing snRNA, rRNA, snoRNA and tRNA sequences. The sequences that mapped to the predicted mature Acythosiphon pisum miRNAs annotated in miRBase were identified as known mature miRNAs by miRDeep2 software. The clean reads from the S. avenae transcriptome have been deposited in the NCBI/SRA database under accession number SRP182781. The Miranda, RNAhybrid, and Target Scan programs were used to predict the target gene of the miRNAs according to the miRNA-binding sites.27-29 Then only the binding sites commonly identified by the three tools were selected for subsequent analysis. The putative miRNA targets were used as queries in searches against the S. avenae transcriptome sequences.
The predicted miRNA target genes were selected for further analysis. The predicted target genes were aligned using the NCBI BLASTX program, and mapping and annotation of the gene sequences were conducted using BLAST2GO.30 The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to further analyze the putative target genes of the miRNAs.
2.5 miRNAs differentially expressed between control and imidacloprid-treated S. avenae
In order to identify the aphid miRNAs that were affected by imidacloprid, the differences in the expression levels of miRNAs, as assessed by miRDeep2, between control and imidacloprid-treated S. avenae were determined. The edgeR software package (3.10.2) obtained from Bioconductor version 3.1 was used to analyze the read counts of the identified miRNAs (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html).31 edgeR is a Bioconductor software package for analyzing the differential expression of digital
gene expression data. The P-values were calculated using the Benjamini-Hochberg method.32 A corrected P-value of 0.05 was set as the default threshold for significance. Normalization of miRNA counts between libraries of the controls and treatments was executed according to the total number of reads across libraries. Normalized expression = Actual miRNA count/Total count of clean reads×106. A fold change in miRNA expression ≥2 with a false discovery rate (FDR) <0.05 was deemed to be significant.
2.6 Quantitative real-time PCR (qPCR) and data analysis
Total RNA was extracted from apterous aphids with TRIzol (Invitrogen) according to the manufacturer's instructions and was then treated with RNase-free DNase I (Takara, Japan). cDNA was synthesized from the total RNA using the PrimeScript™ First-strand cDNA was synthesized from 1.0 μg of total RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara
Biotechnology, Dalian, China). The house-keeping genes actin was used as internal reference gene to determine the expression levels of CYP6A14.
First strand cDNA was synthesized from 2 μg of total RNA using the miScript II RT kit (Qiagen) following the manufacturer’s instructions. The SYBR Green Master Mix (miScript SYBR Green PCR Kit, Qiagen) was used for miRNA expression assays, and qPCR was performed as previously described.33 Relative expression was calculated using the 2−∆∆Ct method.34 U6 snRNA was used to normalize the expression levels of miRNAs as an endogenous control. The primers used are shown in Table 1.
2.7. MiRNA inhibitor feeding in vitro, and the subsequent impacts on CYP6A14 expression and imidacloprid susceptibility
The rearing method and the artificial diet used were the same as previously described.35 A sterilized transparent glass tube with both ends open was used as the feeding device (4 cm in length, 2.5 cm diameter). The artificial diet, 25% sucrose, was put between the two layers of parafilm were sealed or was the parafilm containing the artificial diet used to seal one end of the feeding device, and then healthy apterous adults were transferred to the device; the ends were covered with mesh to prevent the insects from escaping. Each sample contained three replications.
To evaluate modulation of miRNAs and the subsequent impacts on miRNAs and CYP6A14 expression. 3 differentially expressed miRNAs, api-miR-1000, api-miR-316 and api-imR-iab-4 were selected, api-miR-1000 was significantly up-regulated, api-miR-316 and api-imR-iab-4 were significantly down-regulated in the nymphs of S. avenae treated with imidacloprid in comparison with those of the control, and api-miR-316 targeted for CYP6A14. 50 healthy apterous aphids were fed an artificial diet containing miRNA, api-miR-1000, api-miR-316 or api-imR-iab-4 inhibitors at a final concentration of 2.5 mM/L. The NC-inhibitor (a negative control) was used as the control. Following feeding for 24 h, the surviving aphids were collected for RNA extraction. Subsequently, the aphids were collected for qPCR to determine the expression of miRNAs or CYP6A14.
Modulation of miRNAs and the subsequent impacts on imidacloprid susceptibility, 50 healthy apterous adults were fed an artificial diet that contained imidacloprid (at the LC50 concentration, 1.5 mg/L) with an miRNA inhibitor (2.5 mM/L). The NC-inhibitor was used as the control. Three replicates were carried out, and mortality was scored at 48 h. The miRNA inhibitors used were provided by Sangon Biotech Co., Ltd (Shanghai, China).