Figure 4. Altered gene expression in diapausing flies assessed by qPCR suggest endocrine diapause phenotypes.

Relative steady state expression of genes encoding DILPs, InR and AKH in virgin female flies (Canton S), kept for 1–9 weeks under diapause conditions (11°C and 10L:14D), and for one week recovery (R1′) under non-diapause conditions (25°C and 12L:12D) after 3 weeks of diapause. Virgin flies kept for one week at normal conditions (C1) were used as comparison. The expression values were calculated with the 2^−ΔΔCt method relative to that of the 3–6-h old virgin flies (C0) in each assay. Data are presented as means ± S.E.M, n = 4 independent replicates with 10–15 flies in each replicate. Significance of differences from 1 week non-diapause control (1) is indicated as well between groups indicated by connectors, p<0.05, ** p<0.01, *** p<0.001, N.S. not significantly different (ANOVA followed with Tukey test) or alternatively ^# p<0.05, ^## p<0.01 or ^#### p<0.01 (Kruskal–Wallis test followed by pairwise comparisons using Wilcoxon rank sum test). A Compared to 1-week old non-diapausing flies (C1) the dilp2 expression increased significantly after one week of diapause (D1) and remained significantly higher over diapause (D3–D9). After one week of recovery from diapause (R1′) the dilp2 expression decreased significantly back to the C1 level. Interestingly the recently hatched control flies (C0) display a significantly higher dilp2 expression compared to 1-week non-diapausing flies (C1). B Compared to C1 controls dilp3 increased after one week of diapause and remained higher during 9 weeks (D1–D9). After one week of recovery (R1′) from diapause dilp3 decreased back to the control level (C1). The dilp3 expression is significantly higher in recently eclosed flies (C0) than in one week normal controls (C1). C The dilp5 expression showed a profile similar to that of dilp3. D The dilp6 expression was significantly higher throughout diapause than in C1 controls, but with a slight decrease after 6 weeks (D6). After one week of recovery from diapause dilp6 decreased significantly. Recently hatched flies (C0) display higher dilp6 expression than the one week old ones (C1). E The insulin receptor (InR) transcript displayed no significant differences between the treatments. F The Akh mRNA increased drastically after one week in diapause (D1) and peaked after three weeks (D3). The transcript level returned to the one week control (C1) level after recovery from diapause. The Akh expression is not significantly different in recently eclosed flies (C0) and one week normal controls (C1).