Deciphering proteomic signatures of early diapause in Nasonia

Abstract

Insect diapause is an alternative life-history strategy used to increase longevity and survival in harsh environmental conditions. Even though some aspects of diapause are well investigated, broader scale studies that elucidate the global metabolic adjustments required for this remarkable trait, are rare. In order to better understand the metabolic changes during early insect diapause, we used a shotgun proteomics approach on early diapausing and non-diapausing larvae of the recently sequenced hymenopteran model organism Nasonia vitripennis. Our results deliver insights into the molecular underpinnings of diapause in Nasonia and corroborate previously reported diapause-associated features for invertebrates, such as a diapause-dependent abundance change for heat shock and storage proteins. Furthermore, we observed a diapause-dependent switch in enzymes involved in glycerol synthesis and a vastly changed capacity for protein synthesis and degradation. The abundance of structural proteins and proteins involved in protein synthesis decreased with increasing diapause duration, while the abundance of proteins likely involved in diapause maintenance (e.g. ferritins) increased. Only few potentially diapause-specific proteins were identified suggesting that diapause in Nasonia relies to a large extent on a modulation of pre-existing pathways. Studying a diapause syndrome on a proteomic level rather than isolated pathways or physiological networks, has proven to be an efficient and successful avenue to understand molecular mechanisms involved in diapause.

Figure 1. Overlap between proteins/peptides found in non-diapausing and diapausing larvae.

Shown is the overlap of protein/peptide hits for all collected specimens (NDP6, DP6-8) in a 4-way Venn diagram. DP6-8: diapausing larvae collected at days 6–8 (orange, red, and green, respectively). NDP6: non-diapausing larvae collected at day 6 (blue). Hits only identified in the diapause-stages are highlighted in bold.

Figure 2. Heatmap and hierarchical clustering of selected proteins.

This protein expression map (heatmap) visualizes protein abundance differences between individual samples. Proteins shown were selected out of all quantifiable proteins by a Kruskal Wallis test. This procedure resulted in proteins that showed significant (bootstrap corrected p-value cutoff: p≤0.1) differences over the four sample groups (NDP6 and DP6-8). The abundance increases from blue to yellow. Samples were grouped based on Pearson correlation and full linkage clustering. Rows indicate individual proteins, columns indicate individual samples. Comparisons of protein abundances are valid within rows but not within colums. DP6-8: diapausing larvae collected at days 6–8 (orange, red, and green, respectively). NDP6: non-diapausing larvae collected at day 6 (blue).

Figure 3. Abundance changes in putative stress-response proteins during early diapause in Nasonia.

Abundance levels of proteins similar to Ferritin, Ferritin 2 (panels A and B, respectively), HSP90, and LEFL, a member of the HSP20 family (panels C and D, respectively) represented in box plots (median and 25–75% confidence interval). Letters denote significant differences (Mann Whitney U-test, p≤0.05, n = 6 per group). Open circles represent outliers. Spectral count_corr: individual spectral count normalized to total spectral count. DP6-8: diapausing larvae collected at days 6–8 (orange, red, and green, respectively). NDP6: non-diapausing larvae collected at day 6 (blue).

Figure 4. Suggested pathway for glycerol synthesis in Nasonia.

Proteins putatively involved in glycerol production and glycolysis had significantly different abundance levels between groups (Kruskal Wallis and Mann Whitney U-tests, p≤0.05) leading us to suggest this pathway for glycerol synthesis in Nasonia (enzymes in red). Enzymes in blue were detected at higher levels in diapausing stages but did not pass all criteria for quantification. GAPDH: glyceraldehyde-3-phosphate-dehydrogenase, GADH: glycerol-3-phosphate dehydrogenase, PGM: phosphoglucomutase.