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First published online October 5, 2007
Journal of Experimental Biology 210, 3525-3537 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006791
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Search for hepatopancreatic ecdysteroid-responsive genes during the crayfish molt cycle: from a single gene to multigenicity

Assaf Shechter1, Moshe Tom2, Yana Yudkovski2, Simy Weil1, Sharon A. Chang3, Ernest S. Chang3, Vered Chalifa-Caspi4, Amir Berman4,5 and Amir Sagi1,4,*

1 Department of Life Sciences, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel
2 Israel Oceanographic and Limnological Research, Tel-Shikmona, PO Box 8030, Haifa 31080, Israel
3 Bodega Marine Laboratory, University of California-Davis, PO Box 247, Bodega Bay, CA 94923, USA
4 National Institute for Biotechnology in the Negev, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel
5 Department of Biotechnology Engineering, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel


Figure 1
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  		Fig. 1. Changes in gastrolith size, ecdysteroids levels and development of setae during an endocrinologically induced molt cycle in an XO–SG-extirpated male C. quadricarinatus. (A) Digital X-ray imaging of gastrolith growth during a representative endocrinologically induced molt cycle of XO–SG-extirpated male. White arrows point to the gastroliths. Times of endocrine induction and ecdysis are shown by gray arrows. (B) Changes in gastrolith size (squares) determined by MMI (molt mineralization index: gastrolith width as detected by X-ray imaging/carapace length), and circulating ecdysteroids levels (pg µl–1; circles). The x axis is normalized to days from ecdysis. (C) Diagram and light microscope sections of the maxillar exopodite during intermolt (stage C, broken square) and three stages of premolt (D0, D1 and D2). Arrows indicate apolysis regions. Magnification x20.

 

Figure 2
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  		Fig. 2. Changes in gastrolith size during an induced molt cycle in a 20E-injected male C. quadricarinatus. (A) Digital X-ray imaging of the gastrolith in two representative specimens, one injected with 20E (bottom) and the other with carrier (top), which served as control. Images were obtained in the premolt stage on days –13, –6 and –4 relative to the anticipated time of ecdysis; a round metal grid was used for size calibration (10 mm diameter). (B) Upper graph shows the injection regime of 20E, (from a stock solution: 1 µg µl–1 of a saline buffer containing 10% ethanol) injected twice a day; 1000 pg µl–1 was calculated as the maximum physiological level. The injection regime and volume of the carrier were identical in the control group and 20E-injected groups. The x axis is normalized to days from ecdysis. Lower graph shows changes in MMI during the induced molt cycle by repetitive injections of 20E. The x axis is normalized to days from ecdysis. Squares, 20E-injected animal; circles, carrier-injected control.

 

Figure 3
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  		Fig. 3. Relative quantification of the effect of 20E on CqVg expression in vivo and in primary hepatocyte cell culture. (A) Intact and XO–SG-extirpated crayfish were injected daily with either 20E or carrier until they reached ecdysis. They were then sacrificed, and RNA was extracted for evaluation of CqVg expression levels. The control intact crayfish was not subjected to any injection. Different letters indicate statistical significance (P<0.05). (B) A 24 h primary hepatocyte cell culture from an intermolt male was subjected to four different concentrations of 20E: 1, 10, 100 and 1000 pg µl–1. In the carrier sample no 20E was added. The positive control was a 24 h female primary hepatocytes cell culture with no addition of 20E (Female). Different letters indicate statistical significance (P<0.05).

 

Figure 4
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  		Fig. 4. Assembly of the microarray chip and multigene expression patterns related to elevation of ecdysteroids by two methods of endocrine molt induction. (A) Schematic representation of the hybridization design of the experiment showing each of the biological replicates. Arrowhead represents Cy3 labeling, and tail represents Cy5 labeling. XO–SG, premolt crayfish induced by XO–SG removal; 20E, premolt crayfish induced by repetitive 20E injections. The different colors of the circles in each treatment group represent different individuals, whereas in the reference group the same color represents pooling of the samples. (B) Overview of all the clustered genes according to treatments in the experiment. Red, treatment higher than reference; green, reference higher than treatment; yellow, equal expression. Two representative clusters of the ecdysteroid-responsive genes are indicated by boxes: the green box marks the downregulated cluster and the red box the upregulated cluster. (C) Expression scatter plots of all the genes represented on the chip in the two treatments. M, log2-fold change of normalized emission intensity between the treatment and the control; A, average signal in all treatments; green line, the minimum threshold of A at 8.5; red spots, genes with P<0.05, M value >1 or <–1, and A value >8.5.

 

Figure 5
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  		Fig. 5. Gene expression patterns resulting from the two endocrine manipulations out of all the sequenced and assembled genes from the chip, and the gene ontology (GO) pie graph of the putative ecdysteroid-responsive genes. (A) Schematic overview of the experiment showing the overlap between the two manipulations (XO–SG and 20E), which forms the ecdysteroid-responsive group. Upward-pointing arrows indicate upregulated genes; downward-pointing arrows indicate downregulated genes; `unique sequences' are all the sequenced and clustered genes from the chip. In cases where different clones (spots) from the same gene belonged to different sections of the diagram, these genes were counted multiple times. (B) Pie graph of GO of the putative ecdysteroid-responsive genes. These genes showed a significant response during premolt when ecdysteroid titers were elevated in both treatments.

 

Figure 6
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  		Fig. 6. In vitro confirmation of the effects of 20E on ecdysteroid-responsive genes. Real-time RT-PCR relative quantification of two putative ecdysteroid-responsive genes, digestive cysteine proteinase 1 (A) and trypsin (B) in a 24 h hepatopancreatic tissue culture subjected to four different concentrations of 20E (1, 10, 100 and 1000 pg µl–1). Different letters indicate statistical significance (P<0.05).

 

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© The Company of Biologists Ltd 2007