Larval-Transcriptome Dynamics of Ectropis grisescens Reveals Differences in Virulence Mechanism between Two EcobNPV Strains

Abstract

The biological insecticide, Ectropis obliqua nucleopolyhedrovirus (EcobNPV), has been applied to control the major tea-pest Ectropis grisescens. Previously, the virus strain EcobNPV-QF4 showed higher a mortality rate (58.2% vs. 88.2%) and shorter median lethal-time (13.9 d vs. 15.4 d) on E. grisescens than the strain EcobNPV-QV. However, the mechanism of the difference in virulence between the two strains remains unclear. Using the leaf-disc method, we detected the virulence of the two strains on 3^rd-instar larvae, and found that median lethal-dose (LD₅₀) of EcobNPV-QF4 is 55-fold higher than that of EcobNPV-QV (4.35 × 10⁸ vs. 7.89 × 10⁶). Furthermore, fourteen larva transcriptomes of E. grisescens were subsequently sequenced at seven time-points after ingestion of the two virus strains, yielding 410.72 Gb of raw reads. Differential gene-expression analysis shows that 595, 87, 27, 108, 0, 12, and 290 genes were up-regulated in EcobNPV-QF4 at 0, 2, 6, 12, 24, 36 h and 48 h post ingestion (hpi), while 744, 68, 152, 8, 1, 0, 225 were down-regulated. KEGG enrichment showed that when the virus first invades (eats the leaf-discs), EcobNPV-QF4 mainly affects pathways such as ribosome (p-value = 2.47 × 10^-29), and at 48 hpi EcobNPV-QF4, causes dramatic changes in the amino-acid-synthesis pathway and ribosome pathway (p-value = 6.94 × 10^-13) in E. grisescens. Among these, thirteen key genes related to immunity were screened. The present study provides the first ever comprehensive analysis of transcriptional changes in E. grisescens after ingestion of the two strains of EcobNPV.

Keywords: Ectropis grisescens; Ectropis obliqua nucleopolyhedrovirus; transcriptome; virulence difference.

Figure 1

Death dynamics of E. grisescens feeding on leaves treated with EcobNPV-QV (A) and EcobNPV-QF4 (B). Twenty insects in each assay were tested per dose, with four replicates. Larvae feeding on distilled-water-treated leaves were used as control, to calculate the adjusted mortality-rate.

Figure 2

General analyses of host larval expressed-genes responding to EcobNPV-QV vs. EcobNPV-QF4 infection. Genes with |log₂foldchange| >= 1 and padj (corrected p-value) <= 0.05 were recognized as the differentially expressed genes (DEGs). Figures (A–G) represent the volcano plots of DEGs at 0–48 hpi. Red plots are the genes with significant up-regulated differential expression (up), green plots are those with significant down-regulated differential expression (down), and gray plots are the genes with no significant difference in expression (ns).

Figure 3

Validation of the differentially expressed genes between larvae feeding on EcobNPV-QV- and EcobNPV-QF4-infected leaf using qRT-PCR. The GAPDH gene was used as a control, to normalize the gene expression of EcobNPV-QV vs EcobNPV-QF4 with qRT-PCR. All data represent mean ± SEM (n = 3).

Figure 4

KEGG enrichment-analyses of DEGs among different infected time-points (EcobNPV-QF4 vs EcobNPV-QV). KEGG enrichment-analyses of differentially expressed E. grisescens transcripts were first separated into down-regulated unigenes (top portions) and up-regulated unigenes (bottom portions), and then illustrated in EcobNPV-QF4 vs EcobNPV-QV comparison groups in different time (p-value < 0.05).

Figure 5

Expression of immune-related genes in E. grisescens after ingestion with EcobNPV-QV and EcobNPV-QF4. Each square represents each gene, and the color from red to blue indicates the normalized gene-expression with FPKM from large to small. Hpi: hour post ingestion.