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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 Berman^4,5 and Amir Sagi^1,4,*

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

^* Author for correspondence (e-mail: sagia{at}bgu.ac.il)

Accepted 16 July 2007

The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ–sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregulated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle.

Key words: Cherax quadricarinatus, Decapoda, Crustacea, molt staging, gastrolith X-ray imaging, ecdysteroids, X-organ–sinus gland complex, cDNA microarray, hepatopancreas

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