Nutrigenomics in Arma chinensis: transcriptome analysis of Arma chinensis fed on artificial diet and Chinese oak silk moth Antheraea pernyi pupae

Abstract

Background: The insect predator, Arma chinensis, is capable of effectively controlling many pests, such as Colorado potato beetle, cotton bollworm, and mirid bugs. Our previous study demonstrated several life history parameters were diminished for A. chinensis reared on an artificial diet compared to a natural food source like the Chinese oak silk moth pupae. The molecular mechanisms underlying the nutritive impact of the artificial diet on A. chinensis health are unclear. So we utilized transcriptome information to better understand the impact of the artificial diet on A. chinensis at the molecular level.

Methodology/principal findings: Illumina HiSeq2000 was used to sequence 4.79 and 4.70 Gb of the transcriptome from pupae-fed and artificial diet-fed A. chinensis libraries, respectively, and a de novo transcriptome assembly was performed (Trinity short read assembler). This resulted in 112,029 and 98,724 contigs, clustered into 54,083 and 54,169 unigenes for pupae-fed and diet-fed A. chinensis, respectively. Unigenes from each sample's assembly underwent sequence splicing and redundancy removal to acquire non-redundant unigenes. We obtained 55,189 unigenes of A. chinensis, including 12,046 distinct clusters and 43,143 distinct singletons. Unigene sequences were aligned by BLASTx to nr, Swiss-Prot, KEGG and COG (E-value <10(-5)), and further aligned by BLASTn to nt (E-value <10(-5)), retrieving proteins of highest sequence similarity with the given unigenes along with their protein functional annotations. Totally, 22,964, 7,898, 18,069, 15,416, 8,066 and 5,341 unigenes were annotated in nr, nt, Swiss-Prot, KEGG, COG and GO, respectively. We compared gene expression variations and found thousands of genes were differentially expressed between pupae-fed and diet-fed A. chinensis.

Conclusions/significance: Our study provides abundant genomic data and offers comprehensive sequence information for studying A. chinensis. Additionally, the physiological roles of the differentially expressed genes enable us to predict effects of some dietary ingredients and subsequently propose formulation improvements to artificial diets.

Figure 1. Gene Ontology (GO) categories of the unigenes.

Unigenes were annotated in three categories: biological processes (26 sub-categories, 12,428 sequences), cellular components (12 sub-categories, 7,977 sequences), and molecular functions (13 sub-categories, 4,625 sequences).

Figure 2. Clusters of Orthologous Groups (COG) functional classification.

From 22,964 nr hits, 8,066 sequences were classified into 25 COG categories (E-value <1.0⁻⁵).

Figure 3. Expression level and statistics of the differentially expressed genes (DEG) in artificial diet-fed (AD_1) vs. prey-fed (CY_1) treatments.

(A) Expression level of DEG. (B) Statistics of DGE. In total, the expression levels of 54,977 genes were affected by the artificial diet. Of which, 13,872 DEGs had significantly differential expression levels (FDR ≤ 0.001 and |log₂Ratio| ≥ 1). Among them, 10,261 and 3,611 genes were up-regulated and down-regulated, respectively in AD_1 vs. CY_1.

Figure 4. Gene Ontology (GO) categories of the differentially expressed genes (DEG) in artificial diet-fed (AD_1) vs. prey-fed (CY_1) treatments.

In total, 1,912 DEGs of A. chinensis were annotated in three categories: biological processes (26 sub-categories, 4,713 sequences), cellular components (10 sub-categories, 2,853 sequences) and molecular functions (11 sub-categories, 1,725 sequences).

Figure 5. Insulin and mTOR signaling pathways affected by artificial diet feeding.

(A) Insulin signaling pathway. (B) mTOR signaling pathway. Red and green frames indicate genes and enriched functions that were up- and down-regulated in artificial diet-fed (AD_1) and prey-fed (CY_1) treatments.

Figure 6. Verification of differentially expressed genes (DEG) by qRT-PCR.

(A) DEG data in transcriptome analysis. The fold changes of the genes were calculated as the log2 value of each AD_1/CY_1 comparison and are shown on the y-axis. (B) The qRT-PCR analysis of gene expression data. Expression ratios of selected genes in AD-1 compared to CY_1.

Figure 7. Arma chinensis fed with different diets and schematics of the transcriptome sequencing analysis.

(A) Female adults fed with artificial diet (A1) and Chinese oak silk moth pupae (A2); male adults fed with artificial diet (A4) and Chinese oak silk moth pupae (A3). (B) Experiment pipeline of transcriptome. (C) Assembly process of the data.

Figure 8. Pipeline of bioinformatics analysis.