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First published online August 31, 2007
Journal of Experimental Biology 210, 3245-3254 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007740

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Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases in the tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signaling in the molting gland

Sung Gu Lee¹, Brandon D. Bader¹, Ernest S. Chang² and Donald L. Mykles^1,*

¹ Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
² Bodega Marine Laboratory, University of California-Davis, Bodega Bay, CA 94923, USA

View larger version (67K): [in this window] [in a new window]   Fig. 1. Effects of eyestalk ablation on guanylyl cyclase expression in land crab Gecarcinus lateralis tissues. Total RNA from intact and ES-ablated animals was DNase-treated, reverse-transcribed, and PCR-amplified using sequence-specific primers (see Materials and methods). PCR products after 35 cycles were resolved on 2% agarose gels (for Gl-GC-Iß, Gl-GC-III and Gl-EF2) or 10% polyacrylamide gels (for Gl-GC-II), stained with Ethidium Bromide, and quantified by scanning densitometry. (A) Reversed images of gels. (B) Quantitative analysis from triplicate measurements for each tissue and condition (mean ±1 s.e.m., N=3). Both Gl-GC-1ß (0N and 32N isoforms were not distinguished; primers were directed to a common sequence in the C terminus) and Gl-GC-III were expressed at high levels in ES ganglia (EG), gill (Gi), ovary (Ov) and testis (Ts) from intact animals. Tissues from intact animals differed in relative expression of the three Gl-GC-II isoforms. Gl-GC-II(+9) was expressed at varying levels in all tissues and was the dominant isoform in Y-organ (YO), gill, hepatopancreas (HP) and gonad (Ov and Ts). Gl-GC-II(+18) was expressed at highest levels in heart (Ht), claw muscle (CM) and thoracic muscle (TM). Gl-GC-II(+0) was expressed at low levels in most tissues except ES ganglia, in which it was the major isoform. ES ablation had no significant effect on Gl-GC-Iß (GC1), Gl-GC-II (GC2) and Gl-GC-III (GC3) expression in most tissues (B). The only exception was the increased expression of the Gl-GC-II(+18) isoform in claw muscles from 3 days and 7 days post-ESA animals. Gl-EF2 was expressed at similar levels in all tissues. Significant differences between means were determined by one-way ANOVA post-hoc test (Fisher's PLSD); P values are indicated with brackets (a, P<0.034; b, P<0.013; c, P<0.023).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Regression analysis of Gl-GC-II(+18) isoform expression in striated muscles as a function of ecdysteroid concentration in ES-ablated land crabs. Gl-GC-II(+18) was significantly correlated with hemolymph ecdysteroid concentration in claw muscle, but not in thoracic muscle or heart.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (7K): [in this window] [in a new window]   Fig. 4. Effect of 20-hydroxyecdysone (20E) injection on hemolymph ecdysteroid concentration. Intact land crabs were injected with 20E or vehicle (10% ethanol) and sampled at 4 h, 8 h, 12 h and 24 h after injection. Ecdysteroid was quantified with radioimmunoassay. 20E caused a large, transient increase in ecdysteroid concentration in the hemolymph (values are mean ± 1 s.e.m., N=10).

View larger version (27K): [in this window] [in a new window]   Fig. 5. Effects of 20-hydroxyecdysone (20E) injection on expression of guanylyl cyclases in hepatopancreas, testis, claw muscle and thoracic muscle. Intact land crabs were injected with 20E or vehicle (10% ethanol) and tissues collected at 4 h, 8 h and 12 h after injection. Total RNA was DNase-treated, reverse-transcribed and PCR-amplified using sequence-specific primers (see Materials and methods). PCR products after 35 cycles were resolved on 2% agarose gels (for Gl-GC-Iß, Gl-GC-III and Gl-EF2) or 10% polyacrylamide gels (for Gl-GC-II), stained with Ethidium Bromide, and quantified by scanning densitometry. GC expression was normalized to Gl-EF2 (values are mean ± 1 s.e.m., N=3). Significant differences between means, obtained by one-way ANOVA post-hoc test (Bonferroni–Dunn), are indicated with brackets (a, P<0.05; b, P<0.01; c, P<0.002).