Quantitative Proteomic and Transcriptomic Analyses of Metabolic Regulation of Adult Reproductive Diapause in Drosophila suzukii (Diptera: Drosophilidae) Females

Abstract

Diapause is a form of dormancy used by many insects to survive adverse environmental conditions, which can occur in specific developmental stages in different species. Drosophila suzukii is a serious economic pest and we determined the conditions for adult reproductive diapause by the females in our previous studies. In this study, we combined RNA-Seq transcriptomic and quantitative proteomic analyses to identify adult reproductive diapause-related genes and proteins. According to the transcriptomic analysis, among 242 annotated differentially expressed genes in non-diapause and diapause females, 129 and 113 genes were up- and down-regulated, respectively. In addition, among the 2,375 proteins quantified, 39 and 23 proteins were up- and down-regulated, respectively. The gene expression patterns in diapause- and non-diapause were confirmed by qRT-PCR or western blot analysis. The overall analysis of robustly regulated genes at the protein and mRNA levels found four genes that overlapped in the up-regulated group and six genes in the down-regulated group, and thus these proteins/genes may regulate adult reproductive diapause. These differentially expressed proteins/genes act in the citrate cycle, insulin signaling pathway, PI3K-Akt signaling pathway, and amino acid biosynthesis pathways. These results provide the basis for further studies of the molecular regulation of reproductive diapause in this species.

Keywords: Drosophila suzukii; RNA-Seq transcriptomics; qRT-PCR; quantitative proteomics; reproductive diapause.

FIGURE 1

TMT analysis of the differentially expressed proteins data in diapause and non-diapause females. (A) The relative quantitative correlation of the two biological replicates of the proteome. (B) Number of differentially quantified proteins. On the basis of duplicate biological replications analyses, only proteins that changed ≥1.2-fold in relative ratios (p < 0.05) were considered. (C) Enrichment and clustering analysis of the quantitative proteomics data sets based on KEGG pathway database, and the quantitative proteins were divided into four groups (Q1–Q4); two groups for down-regulated (Q1: less than 1/1.2; Q2: 1/1.2 -1) and two groups for up-regulated (Q3: 1-1.2, Q4: more than 1.2). The color index (z-score) is showed in the legend, the red color represents the proteins that were significantly accumulated in KEGG pathway.

FIGURE 2

Transcriptomic analysis of the DEGs data in diapause and non-diapause females. (A) Number of significantly changed annotated DEGs, the conditions for genes was FDR ≤ 0.01 and FC ≥ 2. (B) The distribution of pathways of DEGs annotated in the KEGG data library.

FIGURE 3

Validation of differentially expressed genes/proteins. Thirty DEGs and five proteins were selected. Fifteen up-regulated genes (A) and 15 down-regulated genes (B) by transcriptomic analysis, respectively. Three up-regulated proteins and two down-regulated proteins (C) represent the quantitative proteomic analysis results, respectively. The dark yellow bars represent the qRT-PCR results, presented as the mean ± SE (n = 3), gene ID form http://spottedwingflybase.oregonstate.edu/query. The proteins were separated on a 12% SDS-PAGE gel and immunoblotted with antibodies. α-Tubulin was used as an internal control, each protein sample was analyzed in triplicate (C).

FIGURE 4

The representative MS/MS spectra of some differentially expressed proteins. (A) FK506-binding protein 12 kDa (B) Forkhead box protein O (C) Juvenile hormone acid O-methyltransferase (D) Yolk protein-1.

FIGURE 5

Enrichment and clustering analysis of the quantitative transcriptome and proteome data sets based on GO and KEGG annotations. Quantifiable genes and proteins were classified by gene ontology annotation based on three categories: (A) molecular function; (B) cellular compartment, and (C) biological process. Quantifiable proteins were annotated based on the KEGG pathway database (D). These ratios were classified in six groups, red arrowhead represents up-regulated, green arrowhead represents down-regulated, gray square represents no changed. The p-values were transformed into z-scores prior to hierarchical clustering analysis.

FIGURE 6

Proposed model for adult reproductive diapause regulation in SWD. Arrows indicate stimulation and T-bars indicate suppression. Black and gray indicate the ON and OFF activity states of the genes, respectively.