Fig. 3. Effects of eyestalk ablation (ESA) on guanylyl cyclase and ecdysone receptor expression in the Y-organ of land crabs. Total RNA from intact and ES-ablated animals was DNase-treated, reverse-transcribed and PCR-amplified using sequence-specific primers (see Materials and methods). (A) PCR products after 35 cycles were resolved on a 2% agarose gel (for Gl-GC-Iß, Gl-GC-III and Gl-EF2) or a 10% polyacrylamide gel (for Gl-GC-II) and stained with Ethidium Bromide (reversed images). ESA increased mRNA levels of the two Gl-GC-Iß isoforms (0N and 32N) and Gl-GC-III, but had no effect on mRNA levels of Gl-GC-II(+9), Gl-GC-II(+0) and Gl-EF2. (B) Real-time RT-PCR quantification of Gl-GC-1ß, Gl-GC-II, Gl-GC-III and EcR normalized to Gl-EF2. The Gl-EF2 transcript copy numbers were 3.58x10⁸ at Day 0, 2.15x10⁸ at Day 1, 1.81x10⁸ at Day 3 and 1.56x10⁸ at Day 7 (The amount of cDNA from intact animals was doubled in the PCR to compensate for low mRNA levels of Gl-GC-Iß and Gl-GC-III; see Materials and methods). The data are presented as the fold difference with respect to transcript levels in YOs from intact animals (0 day post-ESA). The transcript copy numbers in intact YOs before normalization were 1.01x10¹⁰ for Gl-GC-Iß, 1.30x10⁸ for Gl-GC-II, 3.47x10¹⁰ for GC-III and 5.00x10⁵ for Gl-EcR. Gl-GC-Iß, Gl-GC-III and Gl-EcR mRNA levels increased tenfold, fourfold and twofold, respectively, by 7 days post-ESA, whereas Gl-GC-II level was not significantly correlated with days post-ESA.