Describing the Diapause-Preparatory Proteome of the Beetle Colaphellus bowringi and Identifying Candidates Affecting Lipid Accumulation Using Isobaric Tags for Mass Spectrometry-Based Proteome Quantification (iTRAQ)

Abstract

Prior to entering diapause, insects must prepare themselves physiologically to withstand the stresses of arresting their development for a lengthy period. While studies describing the biochemical and cellular milieu of the maintenance phase of diapause are accumulating, few studies have taken an "omics" approach to describing molecular events during the diapause preparatory phase. We used isobaric tags and mass spectrometry (iTRAQ) to quantitatively compare the expression profiles of proteins identified during the onset of diapause preparation phase in the heads of adult female cabbage beetles, Colaphellus bowringi. A total of 3,175 proteins were identified, 297 of which were differentially expressed between diapause-destined and non-diapause-destined female adults and could therefore be involved in diapause preparation in this species. Comparison of identified proteins with protein function databases shows that many of these differentially expressed proteins enhanced in diapause destined beetles are involved in energy production and conversion, carbohydrate metabolism and transport, and lipid metabolism. Further hand annotation of differentially abundant peptides nominates several associated with stress hardiness, including HSPs and antioxidants, as well as neural development. In contrast, non-diapause destined beetles show substantial increases in cuticle proteins, suggesting additional post-emergence growth. Using RNA interference to silence a fatty acid-binding protein (FABP) that was highly abundant in the head of diapause-destined females prevented the accumulation of lipids in the fat body, a common product of diapause preparation in this species and others. Surprisingly, RNAi against the FABP also affected the transcript abundance of several heat shock proteins. These results suggest that the identified differentially expressed proteins that play vital roles in lipid metabolism may also contribute somehow to enhanced hardiness to environmental stress that is characteristic of diapause.

Keywords: diapause preparation; fatty acid-binding protein; iTRAQ; lipid metabolism; proteomics; stress resistance.

Figure 1

Schematic life-cycle of the cabbage beetle C. bowringi under laboratory conditions at 25°C. Each segment of the timelines indicates a single day of development (Xue et al., ,; Wang et al., 2004). Zero Represents the day at when female adults newly emerged and that had not yet had the opportunity to feed. One to four represents the days when female adults feed before either beginning reproduction or digging into the soil and entering diapause.

Figure 2

Correlation of the expression patterns between proteins and transcripts as determined by iTRAQ and qRT-PCR on three replicate samples of adult female C. bowringi heads. DD, diapause-destined; NDD, non-diapause-destined. The samples used for iTRAQ and qRTPCR experiments were from distinct batches of heads. ^**P < 0.01, ^*P < 0.05, ns, not significant (t-test).

Figure 3

COG function classification. Peptides that were differentially expressed in the heads of diapause-destined vs. non-diapause-destined female C. bowringi beetles were mapped to protein orthologs and assigned putative functions based on the Clusters of Orthologous Groups of proteins database. Many of the categories of proteins that were up-regulated in diapause-destined females were metabolism and engeretics, as may be expected for individuals packing on additional nutrient stores during the diapause preparatory period.

Figure 4

FABP expression profiles obtained by qRT-PCR on three replicate samples of diapause-destined adult female C. bowringi heads. (A) Spatial expression of FABP in different tissues. (B) Temporal expression of FABP in the fat body after adult eclosion. (C) Temporal expression of FABP in the head after adult eclosion. Day 0 indicates the day on which female adults closed, but had not yet had the opportunity to feed; days 1–5 indicate the number of days in which females fed post-eclosion. Different letters indicate significant difference in the FABP expression levels (P < 0.05, ANOVA followed by LSD test).

Figure 5

Effects of FABP RNAi on FABP transcript abundance (A), fat body morphology (B), lipid droplet accumulation (C) and heat shock protein transcript abundance (D), in diapause-destined adult female C. bowringi. FABP expression in the fat body and head were determined by qRT-PCR on three replicate samples 2 and 4 days after RNAi injection on the day of adult eclosion. Different letters indicate significant difference in FABP expression (P < 0.05, ANOVA followed by LSD test). Fat body morphology was examined in DD females 4 days after injection; CK = control check group, dsGFP = group with dsRNA of green fluorescent protein injection, dsFABP = RNAi treatment group. Cellular lipid droplet accumulation was detected using Oil Red O staining and the relative number of lipid droplets quantified using ROI Manager in ImageJ. The mean was the average of the optical density of 8 randomized areas. ^**P < 0.01, ns = not significant (t-test). Relative transcript abundance of three different heat shock proteins in the fat body was determined 4 days after RNAi injection. ^*P < 0.05 (t-test).