Metabolic and proteomic profiling of diapause in the aphid parasitoid Praon volucre

Abstract

Background: Diapause, a condition of developmental arrest and metabolic depression exhibited by a wide range of animals is accompanied by complex physiological and biochemical changes that generally enhance environmental stress tolerance and synchronize reproduction. Even though some aspects of diapause have been well characterized, very little is known about the full range of molecular and biochemical modifications underlying diapause in non-model organisms.

Methodology/principal findings: In this study we focused on the parasitic wasp, Praon volucre that exhibits a pupal diapause in response to environmental signals. System-wide metabolic changes occurring during diapause were investigated using GC-MS metabolic fingerprinting. Moreover, proteomic changes were studied in diapausing versus non-diapausing phenotypes using a combination of two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry. We found a reduction of Krebs cycle intermediates which most likely resulted from the metabolic depression. Glycolysis was galvanized, probably to favor polyols biosynthesis. Diapausing parasitoids accumulated high levels of cryoprotective polyols, especially sorbitol. A large set of proteins were modulated during diapause and these were involved in various functions such as remodeling of cytoskeleton and cuticle, stress tolerance, protein turnover, lipid metabolism and various metabolic enzymes.

Conclusions/significance: The results presented here provide some first clues about the molecular and biochemical events that characterize the diapause syndrome in aphid parasitoids. These data are useful for probing potential commonality of parasitoids diapause with other taxa and they will help creating a general understanding of diapause underpinnings and a background for future interpretations.

Figure 1. Comparison of metabolite levels in P. volucre mummies.

Quotients of mean content of diapausing (D) over nondiapausing (ND) are shown (i.e. fold change). Red and green bars represent increased and reduced metabolite levels in D mummies respectively. Stars indicate significant difference between D and ND treatments (t-test, P<0.05). A volcano plot is enclosed within this figure; metabolites are ranked according to their statistical P-value (y-axis) and their relative abundance ratio between D and ND (log₂ fold change) (x-axis). Off-centred metabolites are those that vary the most between D and ND phenotypes. Symbols (▴), (▾) and (•) for up-regulated, down-regulated and unaffected metabolites in D mummies respectively. Refer to Table 1 for metabolites abbreviation.

Figure 2. Multivariate analysis (PCA) on metabolomic data.

Panel A illustrates that plotting the first two principal components (PCs) results in a clear-cut separation of diapausing (D) and nondiapausing (ND) metabotypes along PC1. Lines link individuals to their respective centroids (n = 7). Projection of the 48 variables on the correlation circle is shown in panel B.

Figure 3. Representative image of the separation of P. volucre proteins on a 2D-DIGE gel.

On this merged image, the non diapausing group was labeled with Cy3 (green) and diapausing group was labeled with Cy5 (red). Identified proteins showing differential expression level are annotated on the gel with their respective spot number; complete properties of identified proteins are given in Table 2 and Table S1.

Figure 4. Graphical representation of quantitative proteomics data.

Proteins are ranked in a volcano plot according to their statistical P-value (y-axis) and their relative abundance ratio (log₂ fold change) between nondiapausing (ND) and diapausing (D) phenotypes (x-axis). Off-centred spots are those that vary the most between both groups. All matched spots are represented (symbol ×) together with the 30 spots selected for identification (symbol ▪) with mass spectrometry.

Figure 5. DeCyder output of the identified proteins.

Graphs show the normalized spot volumes from four replicate gels for diapausing (D) and nondiapausing (ND) phenotypes, together with three-dimensional fluorescence intensity profiles and corresponding fold changes (D/ND) of the identified spots. Complete properties of identified proteins are given in Table 2 and Table S1.