Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, <i>Spodoptera exigua</i>

<div><p>The beet armyworm, <i>Spodoptera exigua</i> (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of <i>S. exigua</i> representing all stages including eggs, 1^st to 5^th instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4^th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20–94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited “half-ecdysis”. In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in <i>S. exigua</i> and this study provided putative targets for RNAi pest control.</p></div

Summary of beet armyworm transcriptome data sequenced by Illumina Solexa platform.

<p>Summary of beet armyworm transcriptome data sequenced by Illumina Solexa platform.</p

Relative mRNA abundance of nine target genes after injecting siRNAs.

<p>The mRNA levels of target genes were monitored by qRT-PCR at 24, 48, 72, 96 and 120 h after injection. Three insects were collected for each time point. The data for PGCP at 24 h, chitinase 1 at 96 h, and tubulin2 at 120 h are absent because of the unsuccessful RNA purification. Larvae injected with siRNA with a random sequence were used as negative controls. Two housekeeping genes, G3PDH and E2F, were used as multiple internal controls. The qRT-PCR data were analyzed using the delta-delta Ct method. The mRNA abundance of target genes in the negative control was used as the calibrator sample. The mRNA levels of target genes in the siRNA-treated group were relative to the negative control group at the same time point. Error bars indicate standard errors. Statistical significance of differences were analyzed with student T-test (decreasing significance,* P<0.05, **P<0.01; increasing significance, # P<0.05 ).</p

Gene ontology classification of the beet armyworm transcriptome.

<p>Blast2GO was used to analyze the 18,592 contigs identified by Blastx as having significant homology to genes in the NCBI nr database. “Cellular process” and “metabolic process” in biological process, “catalytic activity” and “binding” in molecular function, “cell” and “cell part” in cellular component were the most abundant.</p

Developmental defects in siRNA-treated individuals.

<p>(A) Larvae in the negative control group injected with siRNAs with a random sequence. (B) The “half-ecdysis” in chitinase1-siRNA and chitinase7-siRNA groups. The photo was taken 24 h after siRNA injection. Red arrow indicates the part of larval body still in the old cuticle. This larva could not complete ecdysis and eventually died. (C) The “black body” in arf1-siRNA and arf2-siRNA groups. The photo was taken 72 h after siRNA injection. Red arrow indicates black color in the body.</p

The mortality in RNAi-treated groups at different times after injection.

<p>The mortalities were calculated at 24, 48, 72, 96 and 120 h after injecting siRNAs. The group injected with siRNA with a random sequence was used as the negative control (NC). We did not observe mortality in the NC group. No mortality was observed at 24 h post injection in the chitinase 7 and PGCP siRNA group. Knockdown of eight genes led to significantly high mortality at 72, 96 and 120 h after siRNA injection. All data were analyzed with student T-test (* P<0.05, **P<0.01).</p

Retarded development in RNAi-treated groups.

<p>About 80% of surviving larvae in each siRNA-treated group showed retarded development. The group injected with siRNA with a random sequence was used as the negative control (NC). All photos were taken 48 h after injecting siRNAs. All the treatments are in same condition. To take good photos, we moved the insects to the lid of Petri dishes. However, in some cases, we took the picture directly with diet as the background.</p

Observation of siRNA spread in the larvae after injection by fluorescent microscopy.

<p>Larvae injected with fluorescence labeled chitinase1-siRNA were observed under light (A) and fluorescent microscopy (B), which shows the immediate spread of siRNA to the whole body. The photos were taken five minutes after injection. The controls were un-injected larvae.</p

COG functional classification of the beet armyworm transcriptome.

<p>Orthologous analysis of 18,592 Trinity contigs was performed using the Blastall software against Cluster of Orthologous Groups (COG) database. The clusters “general function prediction only” and “Replication, recombination and repair” were abundant in the beet armyworm transcriptome data.</p

Sequence information of nine candidate genes in <i>S. exigua</i> amplified by RACE strategy.

*<p>Full length</p>**<p>intact CDS/5′UTR/3′UTR.</p