JIA-2019-11

2582 CUI Dong-nan et al. Journal of Integrative Agriculture 2019, 18(11): 2579–2588 them, 175 proteins (representing 1 385 peptides) were differentially expressed between diapause and non- diapause eggs at P <0.05 (≥1.5-fold change, FDR≤0.01) including 84 proteins at P <0.01 (Fig. 2). Among the 175 DEPs, 24 proteins were up--regulated, and 151 were down- regulated (Fig. 3). The 24 significantly up-regulated and the top 20 significantly down-regulated proteins are shown in Tables 2 and 3, respectively. 3.3. GO classification of DEPs Proteins were annotated by the GO database based on their molecular function, cellular component and biological process to further analyze the biological variability of the DEPs (Tu et al. 2015). The most enriched GO terms are shown in Fig. 4. Under the category of biological processes, small molecule metabolic process (19, 10.9%), organic acid metabolic process (12, 6.9%) and carboxylic acid metabolic process (12, 6.9%) were the most represented. Within cellular components, proton-transporting two-sector ATPase complex (4, 2.3%), ribonucleoprotein complex (12, 6.9%) and macromolecular complex (18, 10.3%) were highly represented. For molecular functions, oxidoreductase activity (20, 11.4%), oxidoreductase activity acting on the CH–CH group of donors (4, 2.3%) and acyl-CoA dehydrogenase activity (3, 1.7%) were the most represented. 3.4. KEGG pathway analysis of DEPs In order to determine the biological processes participating in diapause, we used KOBAS 3.0 Software to test the statistical enrichment of DEPs in KEGG pathways (Xie et al. 2011). The 175 DEPs between non-diapause eggs and diapause eggs were mapped to 69 pathways in the KEGG database. But only 40 pathways were substantially enriched ( P <0.05) (Table 4). Metabolic pathways (KEGG: dme01100), carbon metabolism (KEGG: dme01200) and protein processing in endoplasmic reticulum (KEGG: dme04141) were among the top three pathways identified by the pathway analysis. 3.5. Validation by qRT-PCR To detect changes in mRNA levels, four important proteins, hexamerin-like protein 4, JHEH1, cytochrome P450, and HSP20.7, were used for relative quantitative analysis. Results showed the relative expressions of hexamerin- like protein 4, JHEH1, cytochrome P450, and HSP20.7 were higher in non-diapause eggs compared to diapause eggs ( P <0.05; Fig. 5). The trends in relative quantitative expression of JHEH1, cytochrome P450, and HSP20.7 were similar to the proteomics results. However, the relative quantitative expression of hexamerin-like protein 4 was contrary to the proteomics. 4. Discussion Photoperiod and incubation temperature are critical induction factors in facultative diapause of insects, somehow * 0 20 40 60 80 100 16 h L:8 h D 10 h L:14 h D Diapause rate (%) Different photoperiods Fig. 1 Diapause rates of egg produced from females exposed at different photoperiods. * indicates significant differences ( P <0.05) between different photoperiods. 84 (7.69%) 1 090 175 (16.04%) Fig. 2 Statistics of differentially expressed proteins. Out of the 1090 identified proteins, 175 (16.05%) revealed significant differences ( P <0.05) including 84 (7.69%) proteins that were significant at P <0.01. 24 151 0 50 100 150 200 Up-regulated Down-regulated Number of DEPs Fig. 3 Number of differentially expressed proteins (DEPs) in diapause eggs treated at short photoperiod (10 h L:14 h D) as compared to non-diapause eggs (control) treated at long photoperiod (16 h L:8 h D ).