Identifying potential RNAi targets in grain aphid (Sitobion avenae F.) based on transcriptome profiling of its alimentary canal after feeding on wheat plants

Abstract

Background: The grain aphid (Sitobion avenae F.) is a major agricultural pest which causes significant yield losses of wheat in China, Europe and North America annually. Transcriptome profiling of the grain aphid alimentary canal after feeding on wheat plants could provide comprehensive gene expression information involved in feeding, ingestion and digestion. Furthermore, selection of aphid-specific RNAi target genes would be essential for utilizing a plant-mediated RNAi strategy to control aphids via a non-toxic mode of action. However, due to the tiny size of the alimentary canal and lack of genomic information on grain aphid as a whole, selection of the RNAi targets is a challenging task that as far as we are aware, has never been documented previously.

Results: In this study, we performed de novo transcriptome assembly and gene expression analyses of the alimentary canals of grain aphids before and after feeding on wheat plants using Illumina RNA sequencing. The transcriptome profiling generated 30,427 unigenes with an average length of 664 bp. Furthermore, comparison of the transcriptomes of alimentary canals of pre- and post feeding grain aphids indicated that 5490 unigenes were differentially expressed, among which, diverse genes and/or pathways were identified and annotated. Based on the RPKM values of these unigenes, 16 of them that were significantly up or down-regulated upon feeding were selected for dsRNA artificial feeding assay. Of these, 5 unigenes led to higher mortality and developmental stunting in an artificial feeding assay due to the down-regulation of the target gene expression. Finally, by adding fluorescently labelled dsRNA into the artificial diet, the spread of fluorescence signal in the whole body tissues of grain aphid was observed.

Conclusions: Comparison of the transcriptome profiles of the alimentary canals of pre- and post-feeding grain aphids on wheat plants provided comprehensive gene expression information that could facilitate our understanding of the molecular mechanisms underlying feeding, ingestion and digestion. Furthermore, five novel and effective potential RNAi target genes were identified in grain aphid for the first time. This finding would provide a fundamental basis for aphid control in wheat through plant mediated RNAi strategy.

Figure 1

The relative expression levels of differentially expressed genes in grain aphid alimentary canal upon feeding. In total, 5490 genes were found to be differentially expressed among which up-regulated genes were indicated with purple points, whereas down-regulated genes with blue points. The selected 16 genes were indicated by yellow point.

Figure 2

A Venn diagram illustrating shared and unique DEGs annotated in nr, Swissprot, COG, GO and KEGG public databases. Among 5490 DGEs, 3805 annotated in at least one of the public databases, including 3729, 237, 1295, 2322, 733 in nr, Swissprot, COG KEGG and GO databases, respectively.

Figure 3

qRT-PCR validation of the candidate RNAi target genes in grain aphid upon feeding. (*Student’s t-test, n = 3, p < 0.05; **Student’s t-test, n = 3, p < 0.01).

Figure 4

Tissue-specific expression manner of 16 candidate RNAi target genes in grain aphid. Semi-quantitative RT-PCRs were performed using the total RNA from the different tissues of the third instars of grain aphid, such as head, alimentary canal and fat body without alimentary canal.

Figure 5

The effects of different concentration of C002 dsRNA at different time point on the mortality of third instars of grain aphid. The blank artificial diet without dsRNA was used as control. The aphid survival was monitored in an 8 d period. (*Student’s t-test, n = 3, p < 0.05; **Student’s t-test, n = 3, p < 0.01).

Figure 6

The mortality of the third instars of grain aphid feed on artificial diet added with dsRNA of selected candidate RNAi target genes. CK, represented the artificial diet control without dsRNA. The dsRNAs of sixteen candidate target genes along with that of C002 gene were added to the artificial diet at a concentration of 7.5 ng/μl, respectively. The survival of the instars was monitored in an 8 d period. (*Student’s t-test, n = 3, p < 0.05; **Student’s t-test, n = 3, p < 0.01).

Figure 7

The relative expression levels of 5 candidate RNAi target genes upon feeding on artificial diet containing dsRNAs at different time point. The dsRNAs were supplied to the artificial diet at concentration of 7.5 ng/μl. qRT-PCRs were performed using the total RNA from the survived instars at different time point after feeding on artificial diet containing the respective dsRNAs. The relative expression levels of 5 RNAi target genes were monitored at indicated time point in an 8 d period. The expression of Unigene 8273, Unigene 23028 and Unigene 28469 was knocked down significantly after dsRNA feeding at day 6, with the latter two genes completely silenced at day 8. (*Student’s t-test, n=3, p<0.05; **Student’s t-test, n=3, p<0.01).

Figure 8

A systemic RNAi effect in grain aphid observed by using the fluorescent labeled dsRNA added to the artificial diet. The fluorescent labelled dsRNA were added to the artificial diet at a final concentration of 7.5 ng/μl. Fifteen third instar grain aphids were inoculated. (A) 1-2 h later, the fluorescence signal mainly observed in the mouthpart. (B) and (C) 3 h to 5 h later, strong fluorescent signals was mainly centralized in the midgut. (D) In the following time period of artificial diet feeding (6-24 h period), the fluorescent signal began spread around the whole body tissues. From A to D, the left panel of each line was observed under stereo microscope (SM), the middle panel of each line was on bright field (BF) and right panel was on 559 nm (Cy3) under Confocal Fluorescent Microscopy FV1000.