Article pubs.acs.org/jpr
Integrated Proteomic and Metabolomic Analysis of Larval Brain Associated with Diapause Induction and Preparation in the Cotton Bollworm, Helicoverpa armigera Qi Zhang, Yu-Xuan Lu, and Wei-Hua Xu* State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou, China S Supporting Information *
ABSTRACT: Diapause is a developmental arrest that allows an organism to survive unfavorable environmental conditions and is induced by environmental signals at a certain sensitive developmental stage. In Helicoverpa armigera, the larval brain receives the environmental signals for diapause induction and then regulates diapause entry at the pupal stage. Here, combined proteomic and metabolomic differential display analysis was performed on the H. armigera larval brain. Using two-dimensional electrophoresis, it was found that 22 proteins were increased and 27 proteins were decreased in the diapause-destined larval brain, 37 of which were successfully identified by MALDI-TOF/TOF mass spectrometry. RT-PCR and Western blot analyses showed that the expression levels of the differentially expressed proteins were consistent with the 2-DE results. Furthermore, a total of 49 metabolites were identified in the larval brain by GC−MS analysis, including 4 metabolites at high concentrations and 14 metabolites at low concentrations. The results gave us a clue to understand the governing molecular events of the prediapause phase. Those differences that exist in the induction phase of diapause-destined individuals are probably relevant to a special memory mechanism for photoperiodic information storage, and those differences that exist in the preparation phase are likely to regulate accumulation of specific energy reserves in diapause-destined individuals. KEYWORDS: prediapause phase, brain, proteome, metabolome, Helicoverpa armigera
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INTRODUCTION Environmental conditions are not always suitable for insects to sustain continuous development throughout the year, as extremely low or high temperatures, drought or reduced food availability can make their lives harder. As a response, insects have evolved an adaptive strategy whereby developmental arrest (diapause) occurs at a specific stage in their life cycle and enables them to survive unfavorable seasons. Usually, diapause is defined as having three successive phases: prediapause, diapause, and postdiapause, and each phase may comprise several subphases, for example, the prediapause phase is divided into two subphases: the induction phase and the preparation phase.1 The environmental signals are perceived by the insect brain during the induction phase, and the neuroendocrine system serves as the crucial transducer of those ambient signals into the diapause program and regulates specific gene expression to promote metabolic changes during the preparation phase.2 Changes in cuticle composition, the accumulation of greater energy reserves, a high concentration of hemolymph storage proteins, a larger body size, and migration to appropriate places are often seen during the prediapause phase.3 Experiments in Manduca sexta have demonstrated that the brain is the repository of the diapause program.3,4 The © 2011 American Chemical Society
photoperiod is the most important environmental signal for triggering the diapause program, as long or short daylength during the prediapause phase can shift the future developmental fate, and the main receptor of the photoperiod signal lies in the brain.5,6 Although clock genes and their receptors have been identified as involved in the regulation of the circadian rhythm, it is not known how the information generated by the clock is stored and processed by the brain to trigger the diapause program during the prediapause phase. Equally important is determining the diapause decision during the prediapause phase by the identification of early molecular events, but regrettably little is known about this phenomenon.2 The cotton bollworm, Helicoverpa armigera (Noctuidae, Lepidoptera), an agriculturally important pest, undergoes diapause at the pupal stage. H. armigera diapause is induced by a short daylength in the larval stage. The diapause induction phase or photosensitive phase is the fifth larval instar to the early stage of the sixth larval instar, and the diapause preparation phase is the mid-late stage of the sixth larval instar. Therefore, prediapause in H. armigera is defined as the fifthsixth larval instar (unpublished data). After pupation, Received: August 18, 2011 Published: December 13, 2011 1042
dx.doi.org/10.1021/pr200796a | J. Proteome Res. 2012, 11, 1042−1053
Journal of Proteome Research
Article
hybridized once 3−4 years to maintain the diapause character. H. armigera larvae were reared on an artificial diet at 20 °C under a light-dark cycle of 14 L:10 D, resulting in all individuals continue through direct development (nondiapause-destined), or 10 L:14 D, resulting in at least 95% individuals entering pupal diapause (diapause-destined). The principle criterion for pupal diapause in the cotton bollworm is the retention of eye spots in the postgenal region of pupa.24 Larval brains were dissected in ice-cold insect saline containing 0.75% NaCl and stored at −80 °C until use.
individuals incubated under short daylengths will become destined for diapause, and the pupal stage can last at least three months (at 20 °C), while individuals incubated under long daylengths will continue to develop directly into adults for approximately 22−23 days. No obvious phenotypic differences are observed between nondiapause- and diapause-destined larvae. This implies that there are few genes that are differentially expressed between nondiapause- and diapause-destined individuals compared to the large number of genes that function in the regulation of larval growth, development, and metamorphosis. In the nematode Caenorhabditis elegans and the mammalian ground squirrel Spermophilus lateralis, the number of differentially expressed genes in developmental arrest is also low.7,8 Thus, identification of these differentially expressed genes is difficult. Both proteomic and metabolomic techniques are powerful molecular tools. In recent years, proteomic techniques have been successfully used to profile the proteome change in insect growth and development,9−12 and metabolomic techniques have also been widely used in entomology,13,14 although these techniques are seldom utilized to study insect diapause. Using pulse labeling and two-dimensional electrophoresis, Joplin et al. identified diapause specific proteins expressed in the pupal brain of Sarcophaga crassipalpis.15 Li et al. detected an abundance of upregulated heat shock proteins during pupal diapause in S. crassipalpis by proteomic techniques.16 Recently, proteomic analysis of the pupal brain at diapause initiation have been performed in our laboratory, suggesting that differentially expressed proteins act in the metabolic change, stress response, and signal transduction pathways at early pupal stage for diapause initiation.17 Similarly, metabolomic techniques based on gas chromatography−mass spectrometry (GC−MS) have been applied to identify metabolites that were altered in overwintering diapause pupa in S. crassipalpis.18 However, as neither proteomic nor metabolomic analyses have been applied to the molecular events of the prediapause phase, further studies appear to be necessary. More recently, integrated proteomic and metabolomic analysis has been applied to questions in various fields.19−23 In the present study, we performed comparison analyses of the proteome and metabolome in nondiapause- and diapausedestined individuals based on combining two-dimensional electrophoresis and GC−MS analysis to systematically search for the mechanistic basis of the prediapause phase at the molecular level. A total of 49 differentially expressed proteins and 18 altered metabolites were identified in the prediapause phase. Furthermore, gene expression patterns in the two types of individuals were investigated at the mRNA and the protein levels. The results show that changes of proteins and metabolites are involved in the regulation of diapause induction and preparation during the prediapause phase.
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Sample Preparation for Proteomics
About 200−300 larval brains were used as a sample, and three biological replicates were prepared, each in a separate culture. Larval brains were manually homogenized in 250 μL lysis buffer, a solution containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 40 mM dithiothreitol (DTT). Pellets were suspended in 10% TCA with 20 mM DTT. Proteins were precipitated on ice for 1 h. After centrifugation at 12000× g for 15 min at 4 °C, the supernatant was discarded, and protein pellets were washed with cold acetone containing 20 mM DTT three times. Finally, pellets were dried and resuspended in 350 μL lysis buffer together with 0.5% (v/v) IPG buffer (pH 3−10 NL), and 0.001% (w/v) bromophenol blue. Protein concentrations were determined by the Bradford method.25 Two-Dimensional Electrophoresis, Image Acquisition, and Image Analysis
The first dimension was isoelectric focusing (IEF) with immobilized pH gradients (IPGs). The protein samples (340 μL, containing approximately 900 μg proteins) were loaded onto 18 cm immobiline dry strips (pH 3−10, NL; GE Healthcare) and applied by in-gel rehydration in the reswelling tray. The strips were rehydrated overnight at room temperature. The next day, IEF was performed using IPGphor II (GE healthcare) at approximately 20 °C under the following conditions: 200 V for 1 h, 500 V for 1 h, 1000 V for 2 h, gradient from 1000 to 8000 V within 1 h, and 8000 V for 5 h. Prior to the second-dimension, the IPG strips were subjected to equilibration in a buffer containing 6 M urea, 2% (w/v) SDS, 50 mM Tris-HCl (pH 8.8), 30% (w/v) glycerol, and 1% (w/v) DTT for 15 min, followed by a further 15 min equilibration in the same buffer containing 2.5% (w/v) iodoacetamide instead of DTT. Second dimensional electrophoresis was performed on 12% SDS-polyacrylamide gels at 80 V for 4 h, 100 V for 4 h, and 120 V for 2 h. The gels were stained by an MS-compatible silver staining method, in which glutaraldehyde was omitted from the sensitizer. Three independent experiments were performed. Acquisition of gel images was performed by an Image Scanner III (GE Healthcare). All images were imported and analyzed using the software ImageMaster 2D Platinum 7.0 (GE Healthcare) according to the user manual. Based on the detection parameters, spots were automatically detected in each gel, and manual edition was carried out when necessary. The relative volume of a protein spot (% vol) was calculated and used for comparison. Using three biological replicate analyses, only statistically significant (p-value
